Purified antibody composition

ABSTRACT

The invention provides a method for producing a host cell protein-(HCP) reduced antibody preparation from a mixture comprising an antibody and at least one HCP, comprising an ion exchange separation step wherein the mixture is subjected to a first ion exchange material, such that the HCP-reduced antibody preparation is obtained.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.13/532,511, filed on Jun. 25, 2012, which, in turn, is a continuation ofU.S. patent application Ser. No. 12/882,601, filed on Sep. 15, 2010, nowissued as U.S. Pat. No. 8,231,876; which is a division of U.S. patentapplication Ser. No. 11/732,918, filed on Apr. 4, 2007, now issued asU.S. Pat. No. 7,863,426; which claims priority to U.S. provisionalapplication Ser. No. 60/789,725, filed on Apr. 5, 2006 and to U.S.provisional application Ser. No. 60/790,414, filed on Apr. 6, 2006, thecontents of each of which are hereby incorporated in their entirety.

BACKGROUND OF THE INVENTION

Large-scale, economic purification of proteins is increasingly animportant problem for the biotechnology industry. Generally, proteinsare produced by cell culture, using either mammalian or bacterial celllines engineered to produce the protein of interest by insertion of arecombinant plasmid comprising the gene for that protein. Since the celllines used are living organisms, they must be fed with a complex growthmedium, comprising sugars, amino acids, and growth factors, usuallysupplied from preparations of animal serum. Separation of the desiredprotein from the mixture of compounds fed to the cells and from theby-products of the cells themselves to a purity sufficient for use as ahuman therapeutic poses a formidable challenge.

SUMMARY OF THE INVENTION

There is a need for improved methods of obtaining antibody preparationscomprising a reduced amount of host cell protein, including procathepsinL. The invention provides a method for purifying antibodies expressed ina host cell expression system, wherein the resulting antibodypreparation comprises a reduced amount of host cell protein, includingprocathepsin L. The improved method of the invention also includes thedevelopment of reproducible methods of accurately detecting host cellproteins, and a kinetic assay

The invention provides a method for producing a host cell protein-(HCP)reduced antibody preparation from a mixture comprising an antibody andat least one HCP, comprising an ion exchange separation step wherein themixture is subjected to a first ion exchange material, such that theHCP-reduced antibody preparation is obtained. In one embodiment, the ionexchange separation step comprises passing the mixture over the firstion exchange material such that a first eluate having a reduced level ofHCP is obtained. In one embodiment, the method of the invention furthercomprises a second ion exchange separation step wherein the first eluateis subjected to a second ion exchange material such that a firstflowthrough having a reduced level of HCP is obtained. In anotherembodiment, the method of the invention further comprises a hydrophobicinteraction separation step wherein the first flowthrough is subjectedto a first hydrophobic interaction material such that a second eluatehaving a reduced level of HCP is obtained.

In one embodiment of the invention, the ion exchange separation stepcomprises a first ion exchange chromatography step, wherein the mixtureis loaded onto a column comprising the first ion exchange material, suchthat a first eluate having a reduced level of HCP is obtained. In oneembodiment, the invention further comprises a second ion exchangechromatography step comprising loading the first eluate onto a columncomprising a second ion exchange material, such that a first flowthroughis obtained.

In one embodiment, the invention further comprises a hydrophobicinteraction separation step comprising loading the first flowthroughonto a column comprising a first hydrophobic interaction material, suchthat a second eluate is obtained. In one embodiment, the hydrophobicinteraction separation step comprises hydrophobic interactionchromatography. In one embodiment, the hydrophobic interactionchromatography is phenyl sepharose chromatography. In still anotherembodiment, the amount of antibody loaded on to the hydrophobicinteraction material ranges from about 20 to about 40 grams of antibodyper liter of hydrophobic interaction material. In yet anotherembodiment, the amount of antibody loaded on to the hydrophobicinteraction material ranges from about 30 to about 36 grains of antibodyper liter of hydrophobic interaction material.

In one embodiment, the ion exchange chromatography step is cationexchange chromatography. In another embodiment, the cation exchangechromatography is a synthetic methacrylate based polymeric resinattached to a sulfonate group. In still another embodiment, theinvention further comprises washing the ion exchange material with aplurality of wash steps. In one embodiment, the plurality of wash stepscomprises an increase in conductivity. In one embodiment, the ionexchange material is washed with a wash comprising about 40-50% elutionbuffer and about 50-60% water (e.g., water for injection (WFI)). In oneembodiment, the elution buffer is 20 mM Na₂PO₄, 150 mM sodium chloride,pH 7.

In one embodiment, the first eluate is subjected to viral inactivationprior to the first ion exchange chromatography step. In one embodiment,the viral inactivation is achieved through pH viral inactivation (e.g.,lower the pH of the first eluate to thereby inactivate viruses).

In one embodiment of the invention, the second ion exchangechromatography step comprises anion exchange chromatography. In oneembodiment, the anion exchange chromatography is Q sepharosechromatography.

The invention also provides a method for producing a host cellprotein-(HCP) reduced antibody preparation from a mixture comprising anantibody and at least one HCP, wherein the reduced level of HCP isachieved by altering the pH and conductivity of the first eloate suchthat the pH and conductivity of the first eloate is substantiallysimilar to the pit and conductivity of the second ion exchange material.In one embodiment, the pH of the second ion exchange material rangesfrom about 7.7 to about 8.3. In another embodiment, the pH of the firsteluate is altered to range from about 7.7 to about 8.3. In still anotherembodiment, the pH of the first eluate is altered to about 8.0. In oneembodiment, the conductivity of the second ion exchange material rangesfrom about 3.5 mS/cm to about 5.2 mS/cm, or from about 3.5 mS/cm toabout 4.9 mS/cm. In one embodiment, the conductivity of the first eluateis altered to range from about 3.5 mS/cm to about 5.2 mS/cm or fromabout 3.5 mS/cm to about 4.9 mS/cm.

In one embodiment of the invention, the first eluate comprises a rangeof about 90 to about 100 fold less HCP than the mixture as determined bya HCP ELISA. In another embodiment, the first flowthrough comprises arange of about 840 to about 850 fold less HCP than the first eluate asdetermined by a HCP ELISA. In yet another embodiment, the second eluatecomprises a range of about 3 to about 5 fold less HCP than the firstflowthrough as determined by a HCP ELISA.

The invention provides a method for producing a procathepsin L-reducedantibody preparation from a mixture comprising an antibody andprocathepsin L, comprising an ion exchange separation step wherein themixture is subjected to a first ion exchange material, such that theprocathepsin L-reduced antibody preparation is obtained.

In one embodiment, the ion exchange separation step comprises passingthe mixture over the first ion exchange material such that a firsteluate having a reduced level of procathepsin L is obtained. In oneembodiment, the ion exchange separation step comprises a first ionexchange chromatography step, wherein the mixture is loaded onto acolumn comprising the first ion exchange material, such that a firsteluate having a reduced level of procathepsin L is obtained.

In one embodiment, the invention further comprises a second ion exchangeseparation step wherein the first eluate is subjected to a second ionexchange material such that a first flowthrough having a reduced levelof procathepsin L is obtained. In one embodiment, the invention furthercomprises a second ion exchange chromatography step comprising loadingthe first eluate onto a column comprising a second ion exchangematerial, such that a first flowthrough is obtained.

In one embodiment, the invention further comprises a hydrophobicinteraction separalion step wherein the first flowthrough is subjectedto a first hydrophobic interaction material such that a second eluatehaving a reduced level of procathepsin L is obtained. In anotherembodiment, the invention further comprises a hydrophobic interactionseparation step comprising loading the first flowthrough onto a columncomprising a first hydrophobic interaction material, such that a secondeluate is obtained.

In one embodiment, the ion exchange chromatography step is cationexchange chromatography, including, but not limited to a syntheticmethacrylate based polymeric resin attached to a sulfornate group.

In another embodiment, the ion exchange chromatography step furthercomprises washing the ion exchange material with a plurality of washsteps. In one embodiment, the plurality of wash steps comprises anincrease in conductivity. In one embodiment, the ion exchange materialis washed with a wash buffer comprising about 40-50% elution buffer andabout 50-60% water (e.g., water for injection (WFI)). In still anotherembodiment, the elution buffer is 20 mM NaPO₄, 150 mM sodium chloride,pH 7.

In one embodiment of the invention, the first eluate is subjected toviral inactivation prior to ion exchange chromatography step. In oneembodiment, the viral inactivation is achieved through pH viralinactivation (e.g., lowering of the pH of the first eluate to therebyinactive viruses).

In one embodiment, the ion exchange chromatography step comprises anionexchange chromatography. In one embodiment, the anion exchangechromatography is Q sepharose chromatography.

The invention also describes a method wherein the reduced level ofprocathepsin L is attained by altering the pH and conductivity of thefirst eluate such that the pH and conductivity of the first eluate issubstantially similar to the pH and conductivity of the second ionexchange material. In one embodiment, the pH of the second ion exchangematerial ranges from about 7.7 to about 8.3. In another embodiment thepH of the first eluate is altered to range from about 7.7 to about 8.3.In still another embodiment wherein the pH of the first eluate isaltered to about 8.0. In yet another embodiment, the conductivity of thesecond ion exchange material ranges from about 3.5 mS/cm to about 5.2mS/cm, or from about 3.5 mS/cm to about 4.9 mS/cm. In one embodiment,the conductivity of the first eluate is altered to range from about 3.5mS/cm to about 5.2 mS/cm, or from about 3.5 mS/cm to about 4.9 mS/cm.

In one embodiment of the invention, the hydrophobic interactionseparation step comprises hydrophobic interaction chromatography. In oneembodiment, the hydrophobic interaction chromatography is phenylsepharose chromatography. In still another embodiment, the amount ofantibody loaded on to the hydrophobic interaction material ranges fromabout 20 to about 40 grams of antibody per liter of hydrophobicinteraction material. In yet another embodiment, the amount of antibodyloaded on to the hydrophobic interaction material ranges from about 30to about 36 grams of antibody per liter of hydrophobic interactionmaterial.

In one embodiment, the first eluate comprises cathepsin L activityranging from between about 25 to about 60 RFU/s/mg of antibody asmeasured by a cathepsin L kinetic assay.

In another embodiment, the first flowthrough comprises cathepsin Lactivity ranging from between about 0.4 to about 4 RFU/s/mg of antibodyas measured by a cathepsin L kinetic assay.

In still another embodiment, the second eluate comprises cathepsin Lactivity ranging from between about 0.5 to about 1.5 RFU/s/mg ofantibody as measured by a cathepsin L kinetic assay.

In one embodiment of the invention, the level of procathepsin L isreproducibly low.

In a particularly preferred aspect, the invention provides antibodypurification methods in which high amounts of an antibody-HCP mixturecan be loaded onto an ion exchange resin to achieve reduction in HCP inthe mixture. This methodology has the advantage that it can be used withantibody-HCP mixtures that have not been subjected to protein A captureprior to application of the antibody-HCP mixture to the ion exchangeresin. Protein A capture, in which an antibody-HCP mixture is applied toa protein A column such that the antibody binds to protein A and HCPsflow through, typically is used as an initial purification step inantibody purification procedures as a means to remove HCPs. Thus, themethods of the invention are useful for purifying large loads ofantibody-HCP mixtures without the need to carry out a protein Achromatography as an initial step.

Thus, in one embodiment, the invention provides a method for producing ahost cell protein-(HCP) reduced antibody preparation from a mixturecomprising an antibody and at least one HCP, the method comprising:

(a) applying the mixture to a first ion exchange resin in anequilibration buffer, wherein greater than 30 grams of antibody perliter of resin are applied;

(b) washing HCP front the resin with a plurality of wash steps; and

(c) eluting the antibody from the resin with an elution buffer to form afirst eluate,

such that the HCP-reduced antibody preparation is obtained.

In another embodiment, about 35-70 grams of antibody per liter of resinare applied. In yet another embodiment, about 70 grams of antibody perliter of resin are applied. In a preferred embodiment, the mixturecomprising an antibody and at least one HCP is not subjected to proteinA capture (e.g., is not applied to protein A column) prior to applyingthe mixture to the first ion exchange resin.

Preferably, the plurality of wash steps comprises at least a first washand a second wash, wherein there is an increase in conductivity from thefirst wash to the second wash. More preferably, the first wash is withequilibration buffer and the second wash is with a mixture of elutionbuffer and water (e.g., WFI). For example, the mixture of elution bufferand water can comprise about 40-50% elution buffer and about 50-60%water. More preferably, the mixture of elution buffer and water cancomprise about 45% elution buffer and about 55% water. In a preferredembodiment, the elution buffer comprises 20 mM sodium phosphate and 150mM sodium chloride. In this situation, a mixture of elution buffer andwater that is 45% elution buffer and about 55% water is 9 mM sodiumphosphate and 68 mM sodium chloride. In a preferred embodiment, thefirst wash is with an equilibrium buffer comprising 20 mM phosphate, 25mM sodium chloride, the second wash is with a buffer comprising 9 mMphosphate, 68 mM sodium chloride (45% elution buffer, 55% water) and theelution buffer comprises 20 mM sodium phosphate and 150 mM sodiumchloride.

In one embodiment, the method using the first ion exchange resin iscarried out at pH 7. In another embodiment, the method using the firstion exchange resin is carried out at pH 5. In yet another embodiment,the method using the first ion exchange resin is carried out at a pH ina range of about pH 5 to about pH 7, or a range of pH 5 to pH 7. When pH7 is used, preferably about 35 grams of antibody per liter of resin isapplied. When pH 5 is used, preferably about 70 grams of antibody perliter of resin is applied. When a pH in the range of about pH 5 to aboutpH 7 (e.g., pH 5 to pH 7) is used, preferably an amount of antibody fromabout 35 to about 70 grams of antibody per liter of resin (e.g., 35-70grams of antibody per liter of resin) is applied.

In a preferred embodiment, better HCP clearance from the antibody-HCPmixture is achieved (e.g., at pH 5) by loading more antibody onto theresin (e.g., about 70 grams of antibody per liter of resin) than isachieved when less antibody (e.g., about 30 grams of antibody per literof resin) is loaded onto the resin. This is thought to be the result ofdisplacement of HCP from the resin by the antibody when conditions areused at which the binding affinity of the antibody for the resin issignificantly greater than that of HCP for the resin.

Preferably, the first ion exchange resin is a cation exchange resin.Preferably, the cation exchange resin is formed into a column and themixture comprising the antibody and at least one HCP is applied to thecolumn. Preferably, the cation exchange resin comprises a syntheticmethacrylate based polymeric resin attached to a sulfonate group (e.g.,Fractogel S). Alternatively, the cation exchange resin can comprise, forexample, methacrylate or polystyrene based synthetic polymers, silica,highly cross-linked agarose with dextran surface extender, cross-linkedcopolymer of allyl dextran and N. N. methylene bisacryla resins attachedto a sulfonate group, such as sulfonium ions or sulfoethyl.

In another aspect of the invention, after the method using the first ionexchange resin described above is caned out, the method furthercomprises subjecting the first eluate to a viral inactivation step. Forexample, wherein viral inactivation can be achieved by pH viralinactivation to form a virally-inactived preparation (e.g., the firsteluate is subjected to low pH conditions, such as pH of about 3.5, tothereby inactivate viruses). Preferably, the virally-inactivatedpreparation is applied to a second ion exchange resin, wherein, prior toapplying the virally-inactivated preparation to the second ion exchangeresin, pH and conductivity of the virally-inactivated preparation isadjusted to be substantially similar to pH and conductivity of thesecond ion exchange resin. For example, the pH of the second ionexchange resin can be in a range of about pH 7.7 to about pH 8.3 and thepH of the virally-inactivated preparation is adjusted to be in a rangeof about pH 7.7 to about pH 8.3. In another embodiment, the pH of thesecond ion exchange resin can be in a range of about pH 7.8 to about pH8.2 and the pH of the virally-inactivated preparation is adjusted to bein a range of about pH 7.8 to about pH 8.2. More preferably, the pH ofthe second ion exchange resin is about pH 8.0 and the pH of thevirally-inactivated preparation is adjusted to be about pH 8.0.Furthermore, the conductivity of the second ion exchange resin can be ina range of about 3.5 mS/cm to about 5.2 mS/cm and the conductivity ofthe virally-inactivated preparation is adjusted to be in a range ofabout 3.5 mS/cm to about 5.2 mS/cm. Preferably, the conductivity of thesecond ion exchange resin is about 5.0 mS/cm and the conductivity of thevirally-inactivated preparation is adjusted to be about 5.0 mS/cm.

In a preferred embodiment, the second ion exchange resin is an anionexchange resin. For example, the anion exchange resin can be a Qsepharose resin. Preferably, the second ion exchange resin is formedinto a column and the virally-inactivated preparation is applied to thecolumn such that a first flow through is obtained.

In another aspect of the invention, after the first through is obtainedfrom the second ion exchange resin, the first flow through can beapplied to a hydrophobic interaction column such that a second eluate isobtained. In a preferred embodiment, the hydrophobic interaction columnis a phenyl sepharose column. In one embodiment, the first flow throughapplied to the hydrophobic interaction column comprises about 20 toabout 40 grams of antibody per liter of hydrophobic interaction columnmaterial. In another embodiment, the first flow through applied to thehydrophobic interaction column comprises about 30 to about 36 grams ofantibody per liter of hydrophobic interaction column material. Due tothe efficiency of the prior steps in the purification process, it hasbeen found that it is not necessary to subject the second eluate,obtained from the hydrophobic interaction column, to product peakfractionation. Thus, in one embodiment, the second eluate is notsubjected to product peak fractionation.

In a particularly preferred embodiment, the method of the invention forproducing a host cell protein-(HCP) reduced antibody preparation from amixture comprising an antibody and at least one HCP comprises:

(a) applying the mixture to a cation exchange resin in an equilibrationbuffer, wherein the mixture has not been subjected to protein A captureprior to applying to the cation exchange resin and wherein greater than30 grains of antibody per liter of resin are applied;

(b) washing HCP from the cation exchange resin with a plurality of washsteps;

(c) eluting the antibody from the cation exchange resin with an elutionbuffer to form a first eluate;

(d) subjecting the first eluate to a viral inactivation step;

(e) applying the virally-inactivated preparation to an anion exchangeresin to obtain a first flow through; and

(f) applying the first flow through to a hydrophobic interaction columnsuch that a second eluate is obtained;

such that the HCP-reduced antibody preparation is obtained.

In one embodiment, the cation exchange resin is at pH 7 and about 35grams of antibody per liter of resin are applied. In another embodiment,the cation exchange resin is at pH 5 and about 70 grams of antibody perliter of resin are applied. In yet another embodiment, the pH is in arange of about pH 5 to about pH 7 (e.g., pH 5 to pH 7) and an amount ofantibody from about 35 to about 70 grams of antibody per liter of resin(e.g., 35-70 grams of antibody per liter of resin) is applied.

Preferably, the plurality of wash steps comprises washing the resin witha first wash using the equilibration buffer and a second wash using amixture of the elution buffer and water. For example, the mixture ofelution buffer and water can comprise about 40-50% elution buffer andabout 50-60% water (e.g., WFI), more preferably about 45% elution bufferand about 55% water (e.g., WFI). In a preferred embodiment, the elutionbuffer comprises 20 mM sodium phosphate and 150 mM sodium chloride. Inthis situation, a mixture of elution buffer and water that is 45%elution buffer and about 55% water is 9 mM sodium phosphate and 68 mMsodium chloride. In a preferred embodiment, the first wash is with anequilibrium buffer comprising 20 mM phosphate. 25 mM sodium chloride,the second wash is with a buffer comprising 9 mM phosphate, 68 mM sodiumchloride (45% elution buffer, 55% water) and the elution buffercomprises 20 mM sodium phosphate and 150 mM sodium chloride.

Preferably, in the above-described method with steps (a) through (f),prior to applying the virally-inactivated preparation to the anion ionexchange resin (i.e., between steps (d) and (e)), pH and conductivity ofthe virally-inactivated preparation is adjusted to be substantiallysimilar to pH and conductivity of the anion exchange resin. For example,the pH of the second ion exchange resin can be in a range of about pH7.7 to about pH 8.3 and the pH of the virally-inactivated preparation isadjusted to be in a range of about pH 7.7 to about pH 8.3. In anotherembodiment, the pH of the second ion exchange resin can be in a range ofabout pH 7.8 to about pH 8.2 and the pH of the virally-inactivatedpreparation is adjusted to be in a range of about pH 7.8 to about pH8.2. More preferably, the pt of the second ion exchange resin is aboutpH 8.0 and the pH of the virally-inactivated preparation is adjusted tobe about pH 8.0. Furthermore, the conductivity of the second ionexchange resin can be in a range of about 3.5 mS/cm to about 5.2 mS/cmand the conductivity of the virally-inactivated preparation is adjustedto be in a range of about 3.5 mS/cm to about 5.2 mS/cm. Preferably, theconductivity of the second ion exchange resin is about 5.0 mS/cm and theconductivity of the vitally-inactivated preparation is adjusted to beabout 5.0 mS/cm.

In the above-described method with steps (a) through (f), preferably thecation exchange resin is a synthetic methacrylate-based polymeric resinattached to a sulfonate group (e.g., Fractogel), the anion exchangeresin is a Q sepharose resin and the hydrophobic interaction column is aphenyl sepharose column.

Preferably, the first eluate comprises a range of about 90 to about 100fold less HCP than the mixture as determined by a HCP ELISA. Preferably,the first flowthrough comprises a range of about 840 to about 850 foldless HCP than the first eluate as determined by a HCP ELISA. Preferably,the second eluate comprises a range of about 3 to about 5 fold less HCPthan the first flowthrough as determined by a HCP ELISA.

In a particularly preferred embodiment, the method of the invention forproducing a host cell protein-(HCP) reduced antibody preparation from amixture comprising an antibody and at least one HCP comprises:

(a) applying the mixture to a cation exchange resin in an equilibrationbuffer, wherein the cation exchange resin is at pH 7 and about 35 gramsof antibody per liter of resin are applied, or the cation exchange resinis at a pH in a range of pH 5 to pH 7 and about 35 to about 70 grams ofantibody per liter of resin are applied, or the cation exchange resin isat pH 5 and about 70 grams of antibody per liter of resin are applied;

(b) washing HCP from the cation exchange resin with wash stepscomprising a first wash using the equilibration buffer and a second washusing a mixture of an elution buffer and water;

(c) eluting the antibody from the cation exchange resin with the elutionbuffer to form a first eluate;

(d) subjecting the first eluate to a viral inactivation step, whereinviral inactivation is achieved by pH viral inactivation to form avirally-inactived preparation;

(e) applying the virally-inactivated preparation to an anion exchangeresin, wherein, prior to applying the virally-inactivated preparation tothe anion ion exchange resin, pH and conductivity of thevirally-inactivated preparation is adjusted to be substantially similarto pH and conductivity of the anion exchange resin, such that a firstflow through is obtained; and

(f) applying the first flow through to a hydrophobic interaction columnsuch that a second eluate is obtained;

such that the HCP-reduced antibody preparation is obtained.

Preferably, the antibody mixture has not been subjected to protein Acapture prior to applying to the cation exchange resin. Preferably, themixture of elution buffer and water comprises about 40-50% elutionbuffer and about 50-60% water, more preferably about 45% elution bufferand about 55% water (e.g., WFI). In a preferred embodiment, the elutionbuffer comprises 20 mM sodium phosphate and 150 mM sodium chloride. Inthis situation, a mixture of elution buffer and water that is 45%elution buffer and about 55% water is 9 mM sodium phosphate and 68 mMsodium chloride. In a preferred embodiment, the first wash is with anequilibrium buffer comprising 20 mM phosphate, 25 mM sodium chloride,the second wash is with a buffer comprising 9 mM phosphate, 68 mM sodiumchloride (45% elution buffer, 55% water) and the elution buffercomprises 20 mM sodium phosphate and 150 mM sodium chloride. Preferably,the first eluate comprises a range of about 90 to about 100 fold lessHCP than the mixture as determined by a HCP ELISA. Preferably, the firstflowthrough comprises a range of about 840 to about 850 fold less HCPthan the first eluate as determined by a HCP ELISA. Preferably, thesecond eluate comprises a range of about 3 to about 5 fold less HCP thanthe first flowthrough as determined by a HCP ELISA.

In a preferred aspect of any of the above-described purificationmethods, the HCP comprises procathepsin L such that a procathepsinL-reduced antibody preparation is obtained. Preferably, the eluatecomprises cathepsin L activity ranging from between about 25 to about 60RFU/s/mg of antibody as measured by a cathepsin L kinetic assay.Preferably, the first flowthrough comprises cathepsin L activity rangingfrom between about 0.4 to about 4 RFU/s/mg of antibody as measured by acathepsin L kinetic assay. Preferably, the second eluate comprisescathepsin L activity ranging from between about 0.5 to about 1.5RFU/s/mg of antibody as measured by a cathepsin L kinetic assayPreferably, the level of procathepsin L is reproducibly low.

In yet another aspect, the invention pertains to a method for producinga host cell protein-(HCP) reduced antibody preparation from a mixturecomprising an antibody and at least one HCP, the method comprising:

(a) applying the mixture to a cation exchange resin to obtain a firsteluate;

(b) applying the first eluate to an anion ion exchange resin to obtain afirst flow through; and

(c) applying the first flow through to a hydrophobic interaction columnsuch that a second eluate is obtained;

such that the HCP-reduced antibody preparation is obtained.

Preferably, the mixture comprising an antibody and at least one HCP isnot subjected to protein A capture prior to applying the mixture to thefirst ion exchange resin. Preferably, the method further comprisessubjecting the first eluate to a viral inactivation step prior toapplying the first eluate to the anion exchange resin. For example,viral inactivation can be achieved by pH viral inactivation.

Preferably, the cation exchange resin comprises a synthetic methacrylatebased polymeric resin attached to a sulfonate group (e.g., the cationexchange resin can be a Fractogel S column). For example, a Fractogel Scolumn can be equilibrated with an equilibration buffer comprising 20 mMsodium phosphate, 25 mM sodium chloride, the mixture can be applied tothe column, the column can be at least washed once with equilibrationbuffer and the first eluate can be obtained by eluting with an elutionbuffer comprising 20 mM sodium phosphate, 150) mM sodium chloride.

Preferably, the anion exchange resin is a Q sepharose column. Forexample, a Q sepharose column can be equilibrated with an equilibrationbuffer comprising 25 mM trolamine, 40 mM sodium chloride, pH 7.6.

Preferably, the hydrophobic interaction column is a phenyl sepharosecolumn. For example, a phenyl sepharose column can be equilibrated withan equilibriation buffer comprising 20 mM sodium phosphate, 1.1 M(NH₄)₂SO₄, pH 7, the first flowthrough can be applied to the column, thecolumn can be at least washed once with equilibration buffer and thesecond eluate can be obtained by performing a salt step-gradient to 11mM sodium phosphate, 0.625 M (NH₄)₂SO₄, pH 7.0.

Preferably, pH viral inactivation is achieved by maintaining the firsteluate at pH 3.5 for approximately one hour.

In yet another aspect, the invention pertains to a method for producinga host cell protein-(HCP) reduced adalimumab preparation from a mixturecomprising adalimumab and at least one HCP, the method comprising:

(a) applying the mixture to a cation exchange resin, wherein the mixtureis not subjected to protein A capture prior to applying the mixture tothe first ion exchange resin, to obtain a first eluate;

(b) subjecting the first eluate to pH viral inactivation to obtain avirally inactivated preparation;

(c) applying the virally inactivated preparation to an anion ionexchange resin to obtain a first flow through; and

(c) applying the first flow through to a hydrophobic interaction columnsuch that a second eluate is obtained;

such that the HCP-reduced adalimumab preparation is obtained.

Preferably, the cation exchange resin is a Fractogel S column, the anionexchange resin is a Q sepharose column and the hydrophobic interactioncolumn is a phenyl sepharose column. For example, a Fractogel S columncan be equilibrated with an equilibration buffer comprising 20 mM sodiumphosphate, 25 mM sodium chloride, the mixture can be applied to thecolumn, the column can be at least washed once with equilibration bufferand the first eluate can be obtained by eluting with an elution buffercomprising 20 mM sodium phosphate, 150 mM sodium chloride. Also forexample, a Q sepharose column can be equilibrated with an equilibrationbuffer comprising 25 mM trolamine, 40 mM sodium chloride, pH 7.6. Alsofor example, a phenyl sepharose column can be equilibrated with anequilibration buffer comprising 20 mM sodium phosphate, 1.1 M (NH₄)₂SO₄,pH 7, the first flowthrough can be applied to the column, the column canbe at least washed once with equilibration buffer and the second eluatecan be obtained by performing a salt step-gradient to 11 mM sodiumphosphate, 0.625 M (NH₄)₂SO₄, pH 7.0. Also for example, pH viralinactivation can be achieved by maintaining the first eluate at pH 3.5for approximately one hour.

With respect to all of the above-described purification methods, in apreferred embodiment of the invention, the antibody is an anti-tumornecrosis factor-alpha (TNFα) antibody, or antigen-binding portionthereof. In one embodiment, the anti-TNFα antibody, or antigen-bindingportion thereof, is a chimeric antibody, a humanized antibody or amultivalent antibody. In one embodiment, the anti-TNFα antibody, orantigen-binding portion thereof, is infliximab or golimumab.

In another embodiment, the anti-TNFα□ antibody, or antigen-bindingportion thereof, is a human antibody. In one embodiment, the anti-TNFα□antibody, or antigen-binding portion thereof, is an isolated humanantibody that dissociates from human TNFα with a K_(d) of 1×10⁻⁸ M orless and a K_(off) rate constant of 1×10⁻³ s⁻¹ or less, both determinedby surface plasmon resonance, and neutralizes human TNFα cytotoxicity ina standard in vitro L929 assay with an IC₅₀ of 1×10⁻⁷ M or less.

In another embodiment, the anti-TNFα antibody, or antigen-bindingportion thereof, is an isolated human antibody with the followingcharacteristics:

a) dissociates from human TNFα with a K_(off) rate constant of 1×10⁻³s⁻¹ or less, as determined by surface plasmon resonance;

b) has a light chain CDR3 domain comprising the amino acid sequence ofSEQ ID NO: 3, or modified from SEQ ID NO: 3 by a single alaninesubstitution at position 1, 4, 5, 7 or 8 or by one to five conservativeamino acid substitutions at positions 1, 3, 4, 6, 7, 8 and/or 9;

c) has a heavy chain CDR3 domain comprising the amino acid sequence ofSEQ ID NO: 4, or modified from SEQ ID NO: 4 by a single alaninesubstitution at position 2, 3, 4, 5, 6, 8, 9, 10 or 11 or by one to fiveconservative amino acid substitutions at positions 2, 3, 4, 5, 6, 8, 9,10, 11 and/or 12.

In still another embodiment, the anti-TNFα antibody, or antigen-bindingportion thereof, is an isolated human antibody with a light chainvariable region (LCVR) comprising the amino acid sequence of SEQ ID NO:1 and a heavy chain variable region (HCVR) comprising the amino acidsequence of SEQ ID NO: 2

In yet another embodiment, the anti-TNFα antibody, or antigen-bindingportion thereof, is adalimumab.

The invention provides an antibody preparation which is substantiallyfree of HCP as measured by a HCP ELISA produced using any of the methodsof the invention. The invention also provides a pharmaceuticalcomposition comprising an HCP-reduced antibody preparation producedusing any of the methods of the invention, and a pharmaceuticallyacceptable carrier.

The invention includes a pharmaceutical composition comprising anantibody an HCP-reduced antibody, wherein the level of HCP comprises nogreater than about 70 ng of HCP per mg of antibody as measured by a HCPELISA, and a pharmaceutically acceptable carrier. In one embodiment, thelevel of HCP comprises no greater than about 1.3 ng of HCP per mg ofantibody as measured by a HCP ELISA. In another embodiment, the level ofHCP comprises no greater than about 5 ng of HCP per mg of antibody asmeasured by a HCP ELISA.

The invention provides a composition comprising an antibody, whereinsaid composition has no detectable level of HCP as determined by a HCPELISA assay.

The invention also provides an antibody preparation which issubstantially free of procathepsin L produced using any of the methodsdescribed herein. The invention also includes a pharmaceuticalcomposition comprising a procathepsin L-reduced antibody preparationproduced using any of the methods described herein, and apharmaceutically acceptable carrier.

The invention provides a pharmaceutical composition comprising anantibody a procathepsin L-reduced antibody and a pharmaceuticallyacceptable carrier, wherein the level of procathepsin L is no greaterthan a cathepsin activity of about 3.0 RFU/s/mg of antibody.

With respect to all of the above-described antibody preparations andpharmaceutical compositions, preferably the antibody is an anti-tumornecrosis factor-alpha (TNFα) antibody, or antigen-binding portionthereof. In one embodiment, the anti-TNFα antibody, or antigen-bindingportion thereof, is an antibody selected from the group consisting ofhumanized, chimeric or multivalent. In one embodiment, the anti-TNFαantibody, or antigen-binding portion thereof, is infliximab orgolimumab.

In another embodiment, the anti-TNFα□ antibody, or antigen-bindingportion thereof, is a human antibody. In one embodiment, the anti-TNFα□antibody, or antigen-binding portion thereof, is an isolated humanantibody that dissociates from human TNFα with a K_(d) of 1×10⁻⁸ M orless and a K_(off) rate constant of 1×10⁻³ s⁻¹ or less, both determinedby surface plasmon resonance, and neutralizes human TNFα cytotoxicity ina standard in vitro L929 assay with an IC₅₀ of 1×10⁻⁷ M or less.

In another embodiment, the anti-TNFα□ antibody, or antigen-bindingportion thereof, is an isolated human antibody with the followingcharacteristics:

a) dissociates from human TNFα with a K_(off) rate constant of 1×10³ s⁻¹or less, as determined by surface plasmon resonance;

b) has a light chain CDR3 domain comprising the amino acid sequence ofSEQ ID NO: 3, or modified from SEQ ID NO: 3 by a single alaninesubstitution at position 1, 4, 5, 7 or 8 or by one to five conservativeamino acid substitutions at positions 1, 3, 4, 6, 7, 8 and/or 9;

c) has a heavy chain CDR3 domain comprising the amino acid sequence ofSEQ ID NO: 4, or modified from SEQ ID NO: 4 by a single alaninesubstitution at position 2, 3, 4, 5, 6, 8, 9, 10 or 11 or by one to fiveconservative amino acid substitutions at positions 2, 3, 4, 5, 6, 8, 9,10, 11 and/or 12.

In still another embodiment, the anti-TNFα antibody, or antigen-bindingportion thereof, is an isolated human antibody with a light chainvariable region (LCVR) comprising the amino acid sequence of SEQ ID NO:1 and a heavy chain variable region (HCVR) comprising the amino acidsequence of SEQ ID NO: 2

In yet another embodiment, the anti-TNFα antibody, or antigen-bindingportion thereof, is adalimumab.

The invention includes a method of treating a disorder in which TNFαactivity is detrimental comprising administering to a human subject apharmaceutical compositions comprising an antibody obtained using any ofthe methods of the invention. In one embodiment, the preparation isadministering to the human subject over a prolonged period of time. Inone embodiment, the prolonged period of time includes at least about 3months, at least about 4 months or at least about 5 months.

In one embodiment, the disorder in which TNFα activity is detrimental isselected from the group consisting of an autoimmune disorder, anintestinal disorder, and a skin disease. In one embodiment, theautoimmune disorder is selected from the group consisting of rheumatoidarthritis, rheumatoid spondylitis, osteoarthritis, gouty arthritis, anallergy, multiple sclerosis, psoriatic arthritis, autoimmune diabetes,autoimmune uveitis, nephrotic syndrome, and juvenile rheumatoidarthritis. In another embodiment, the intestinal disorder is Crohn'sdisease. In still another embodiment, the skin disease is psoriasis.

In one embodiment, the pharmaceutical composition is administering incombination with an additional therapeutic agent. In one embodiment, theadditional therapeutic agent is methotrexate.

The invention includes a method of treating a disorder in which TNFαactivity is detrimental comprising administering to a human subject thepharmaceutical composition comprising an antibody obtained using any ofthe methods of the invention. In one embodiment, the preparation isadministering to a human subject over a prolonged period of time. In oneembodiment, the prolonged period of time includes at least about 3months, at least about 4 months or at least about 5 months. In oneembodiment, the disorder in which TNFα activity is detrimental isselected from the group consisting of an autoimmune disorder, anintestinal disorder, and a skin disease. In one embodiment, theautoimmune disorder is selected from the group consisting of rheumatoidarthritis, rheumatoid spondylitis, osteoarthritis, gouty arthritis, anallergy, multiple sclerosis, psoriatic arthritis, autoimmune diabetes,autoimmune uveitis, nephrotic syndrome and juvenile rheumatoidarthritis. In one embodiment, the intestinal disorder is Crohn'sdisease. In one embodiment, the skin disease is psoriasis.

In one embodiment, the pharmaceutical composition is administered incombination with an additional therapeutic agent. In one embodiment, theadditional therapeutic agent is methotrexate.

The invention provides an article of manufacture comprising a packagingmaterial, adalimumab, and a label or package insert contained within thepackaging material indicating that the adalimumab formulation comprisesno greater than about 70 ng of HCP per mg of adalimumab. In oneembodiment, the about 70 ng of HCP per mg of adalimumab is measured by aHCP ELISA.

The invention also provides an article of manufacture comprising apackaging material, adalimumab, and a label or package insert containedwithin the packaging material indicating that the adalimumab formulationcomprises no greater than about 13 ng of HCP per mg of adalimumab. Inone embodiment, the about 13 ng of HCP per mg of adalimumab is measuredby a HCP ELISA.

The invention includes an article of manufacture comprising a packagingmaterial, adalimumab, and a label or package insert contained within thepackaging material indicating that the adalimumab formulation comprisesno greater than about 5 ng of HCP per mg of adalimumab. In oneembodiment, the about 5 ng of HCP per mg of adalimumab is measured by aHCP ELISA.

The invention includes an article of manufacture comprising a packagingmaterial, adalimumab, and a label or package insert contained within thepackaging material indicating that the adalimumab formulation comprisesno greater a level of procathepsin L than that indicated by a cathepsinL activity of about 3.0 RFU/s/mg adalimumab. In one embodiment,cathepsin L activity is measured by a cathepsin L kinetic assay.

The invention further provides a kinetic assay for determining theamount of procathepsin L in a material derived from a mammalian cellexpression system comprising contacting the material derived from amammalian cell expression system with an enzyme to process procathepsinL to an active cathepsin L form, such that a cathepsin L sample isobtained; contacting the cathepsin L sample with a substrate forcathepsin L; and determining the cathepsin L activity in the cathepsin Lsample as an indication of the amount procathepsin L in the materialderived from the mammalian cell expression system. In one embodiment,the mammalian cell expression system is Chinese Hamster Ovary (CHO)cells. In another embodiment, the enzyme to process procathepsin L is anendopeptidase. In still another embodiment, the substrate for cathepsinL comprises a label. In still another embodiment, the label is afluorescent agent. In one embodiment, the fluorescent agent comprises afluorescent 7-amino-4-methyl coumarin (AMC) group. In one embodiment,the substrate for cathepsin L comprises Z-leucine-arginine. In stillanother embodiment, the Z-leucine-arginine comprises an AMC group.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B show the typical elution profiles for the Fractogel Schromatography step for each process, including the process of theinvention (FIG. 1A) and process A (FIG. 1B).

FIGS. 2A and 2B show a comparison of the flow-through wash profile of QSepharose FF chromatography step, including the process of the invention(FIG. 2A) and process A (FIG. 2B).

FIGS. 3A and 3B show a comparison of the elution profile of PhenylSepharose HP chromatography step, including process B (FIG. 3A) andprocess A (FIG. 3B).

FIG. 4 shows a graphic depiction of a stepwise reduction in procathepsinL for the average process B (diamond shape) and average process A(square shape).

FIG. 5 shows a graphic depiction of the stepwise reduction in HCP forthe average process B (diamond shape) and process A (square shape).

FIG. 6 shows that kinetic readings of activated in-process samplesindicated the linear relationship of reaction time versus fluorescentsignal.

DETAILED DESCRIPTION OF THE INVENTION I. Definitions

In order that the present invention may be more readily understood,certain terms are first defined.

The term “mixture”, as used herein, refers to a material havingviscosity which is to be purified comprising at least one antibody ofinterest which is sought to be purified from other substances which mayalso be present. Mixtures can, for example, be aqueous solutions,organic solvent systems, or aqueous/organic solvent mixtures orsolutions. The mixtures are often complex mixtures or solutionscomprising many biological molecules (such as proteins, antibodies,hormones, and viruses), small molecules (such as salts, sugars, lipids,etc.) and even particulate matter. While a typical mixture of biologicalorigin may begin as an aqueous solution or suspension, it may alsocontain organic solvents used in earlier separation steps such assolvent precipitations, extractions, and the like. Examples of mixturesthat may contain valuable biological substances amenable to thepurification by various embodiments the present invention include, butare not limited to, a culture supernatant from a bioreactor, ahomogenized cell suspension, plasma, plasma fractions, and milk.

By “purifying” an antibody from a mixture comprising the antibody andone or more substances is meant increasing the degree of purity of theantibody in the mixture by removing (completely or partially) at leastone substance from the composition. The substance may be an impurity orcontaminant, such as, but not limited to, a host cell protein (HCP).

The term “host cell protein(s)” or “HCP(s)” refers to proteins in themixture that are different from the antibody of interest and typicallyoriginate from the source of the antibody production, HCPs are desirablyexcluded from the final antibody preparation.

The term “reduced” refers to the lessening or diminishing the amount ofa substance. A reduced preparation includes a preparation which has lessof a substance, such as HCPs or procathepsin L, relative to an initialamount. In one embodiment, the substance is an impurity or contaminant.In one embodiment, the term “reduced” means substantially less of thesubstance. In another embodiment, the term “reduced” means no amount ofthe substance. In one embodiment, no amount of a substance includes “nodetectable amount” using assays described herein.

The term “substantially free” includes no amount of a substance, but canalso include a minimal amount of a substance. In one embodiment, noamount of a substance includes “no detectable amount” using assaysdescribed herein.

The term “host cell protein-(HCP) reduced” refers to a composition,including, but not limited to, an eluate, an preparation, a flowthrough,comprising an antibody and a lessened or diminished amount of HCP(s)following one or more purification steps. In one embodiment, the term“HCP-reduced” means substantially less of the HCP(s) in the compositioncomprising an antibody. In another embodiment, the term “HCP-reduced”means no amount of the HCP(s) in the composition comprising an antibody.In one embodiment, the term “HCP-reduced” means no detectable amountusing assays described herein in the composition comprising an antibody.

The term “procathepsin L-reduced” refers to a composition, including,but not limited to, an eluate, an preparation, a flowthrough, comprisingan antibody and a lessened or diminished amount of procathepsin Lfollowing one or more purification steps. In one embodiment, the term“procathepsin L-reduced” means substantially less of the HCP(s) in thecomposition comprising an antibody. In another embodiment, the term“procathepsin L-reduced” means no amount of the HCP(s) in thecomposition comprising an antibody. In one embodiment, the term“procathepsin L-reduced” means no detectable amount using assaysdescribed herein in the composition comprising an antibody.

The term “reproducibly low” refers to an ability to consistently achievea lessened or diminished amount, such as an ability to achieve alessened or diminished amount at least 80% of the time, more preferablyat least 90% of the time, more preferably at least 95% of the time andeven more preferably at least 98% of the time.

The term “ion exchange separation step” refers to a step where undesiredsubstances or impurities, e.g., HCPs or procathepsin L, are set apartfrom an antibody of interest based on differences in the ionicinteractions of the antibody of interest and the undesired substancewith a charged material. An example of an ion exchange separation stepincludes, but is not limited to, ion exchange chromatography, includinganion exchange chromatography and cation exchange chromatography.

“Ion exchange material” refers to an ionic material which is used as thebasis for the separation of the undesired substances or impurities,e.g., HCPs or procathepsin L, from the antibody. Examples of ionexchange materials include anionic and cationic resins.

“Cation exchange material” refers to an ion exchange resin withcovalently bound negatively charged ligands, and which thus has freecations for exchange with cations in a solution with which the resin iscontacted. A wide variety of cation exchange resins are known in theart, for example, those wherein the covalently hound groups arecarboxylate or sulfonate. Commercially available cation exchange resinsinclude CMC-cellulose, SP-Sephadex™, and Fast S-Sepharose™ (the lattertwo being commercially available from Pharmacia).

“Anion exchange material” refers to an ion exchange resin withcovalently bound positively charged groups, such as quaternary aminogroups. Commercially available anion exchange resins include DEAEcellulose, TMAE, QAE Sephadex™, and Fast Q Sepharose™ (the latter twobeing commercially available from Pharmacia).

By “binding” a molecule to an ion exchange material is meant exposingthe molecule to the ion exchange material under appropriate conditions(pH/conductivity) such that the molecule is reversibly immobilized in oron the ion exchange material by virtue of ionic interactions between themolecule and a charged group or charged groups of the ion exchangematerial.

The term “hydrophobic interaction step” refers to a step where undesiredsubstances, e.g., HCPs or procathepsin L, are set apart from an antibodyof interest based on the differences in the hydrophobic interactions ofthe antibody of interest and the undesired substance with a hydrophobicmaterial.

The term “hydrophobic interaction material” refers to a hydrophobicmaterial which is used as the basis for the separation of the undesiredsubstances, e.g., HCPs or procathepsin L, and the antibody. Examples ofhydrophobic interaction materials include hydrophobic ligands such asalkyl groups having from about 2 to about 8 carbon atoms, or aryl groupssuch as phenyl.

The term “washing” or “wash step” includes passing an appropriate bufferthrough or over a given material, e.g., ion exchange material orhydrophobic interaction material.

The term “plurality of wash steps” includes more than one successivewash steps The successive buffers may have varying properties such aspH, conductivity, solvent concentration, etc., designed to dissociateand remove varying types of impurities that are non-specificallyassociated with the given material. e.g., ion exchange material orhydrophobic interaction material. In one embodiment, the plurality ofwash steps includes an intermediate wash, further comprising about40-50% elution buffer.

To “elute” a molecule (e.g. antibody or contaminant substance) from amaterial is meant to remove the molecule there from by altering thebuffer surrounding the material and thereby decreasing the interactionof the molecule and the material. In one embodiment, an antibody iseluted from an ion exchange column wherein the buffer competes with theantibody for the charged sites on the ion exchange material.

The term “eluate” refers to liquid comprising the molecule, (e.g.antibody or contaminant substance) which was obtained subsequent to thebinding of the antibody of interest to a chromatography material andaddition of an elution buffer to dissociate the antibody. Eluates may bereferred to with respect to the step in the purification process. Forexample, the term “first eluate” refers to the eluate from the firstchromatographic step, the term “second eluate” refers to the eluate fromthe second chromatographic step, etc.

The tem “flowthrough” refers to a liquid comprising a molecule (e.g.antibody or contaminant substance) which was obtained by passing amixture comprising the molecule over a chromatography material such thatthe molecule passes over the material without binding.

A “buffer” refers to a substance which, by its presence in solution,increases the amount of acid or alkali that must be added to cause unitchange in pH. A buffered solution resists changes in pH by the action ofits acid-base conjugate components. Buffered solutions for use withbiological reagents are generally capable of maintaining a constantconcentration of hydrogen ions such that the pH of the solution iswithin a physiological range. Traditional buffer components include, butare not limited to, organic and inorganic salts, acids and bases.Exemplary buffers for use in purification of biological molecules (e.g.,antibodies) include the zwitterIonic or “Good” Buffers, see e.g., Goodet al. (1966) Biochemistry 5:467 and Good and Izawa (1972) MethodsEnzymol. 24:62. Exemplary buffers include but are not limited to TES,MES, PIPES, HEPES, MOPS, MOPSO, TRICINE and BICINE.

“Wash buffer” as used herein all refer herein to the substance used tocarry away impurities from the given material, e.g., ion exchangematerial or hydrophobic interaction material, to which the antibody isbound.

The “elution buffer” refers to a substance that is used to dissociatethe antibody from the given material, e.g., ion exchange material orhydrophobic interaction material, after it has been washed with one ormore wash substances. The elution buffer acts to dissociate theantibody. Typical elution substances are well known in the art and mayhave higher concentrations of salts, free affinity ligands or analogs,or other substances that promote dissociation of the target substance,e.g., antibody from the given material. The conductivity and/or pH ofthe elution buffer is/are such that the antibody is eluted from the ionexchange or hydrophobic interaction material.

The term “conductivity” refers to the ability of an aqueous solution toconduct an electric current between two electrodes. In solution, thecurrent flows by ion transport. Therefore, with an increasing amount ofions present in the aqueous solution, the solution will have a higherconductivity. The unit of measurement for conductivity is mmhos (mS/cm),and can be measured using a conductivity meter sold, e.g., by Orion. Theconductivity of a solution may be altered by changing the concentrationof ions therein. For example, the concentration of a buffering agentand/or concentration of a salt (e.g. NaCl or KCl) in the solution may bealtered in order to achieve the desired conductivity. In one embodiment,the salt concentration of a wash buffer or any other aqueous solutionused in chromatography is modified to achieve the desired conductivity.

The “pI” or “isoelectric point” of a polypeptide, such as an antibody,refers to the pH at which the polypeptide's positive charge balances itsnegative charge, pI can be calculated from the net charge of the aminoacid residues of the polypeptide or can be determined by isoelectricfocusing.

The term “viral inactivation” includes rendering a virus contained inthe mixture nonfunctional or removing a virus from the mixture to bepurified. The virus may originate from the source of antibodyproduction, downstream processing steps or manufacturing conditions.Methods of rendering a virus nonfunctional or removing a virus includeheat activation, pH inactivation, chemical inactivating agents, etc. Theterm “pH viral inactivation” includes subjecting a virus to a pHsufficient to render the virus nonfunctional.

The term “human TNFα” (abbreviated herein as hTNFα, or simply hTNF), asused herein, is intended to refer to a human cytokine that exists as a17 kI) secreted form and a 26 kD membrane associated form, thebiologically active form of which is composed of a trimer ofnoncovalently bound 17 kD molecules. The structure of hTNFα is describedfurther in, for example, Pennica. D., et al. (1984) Nature 312:724-729;Davis, J. M., et al. (1987) Biochemistry 26:1322-1326; and Jones, E. Y.,et al. (1989) Nature 338:225-228. The term human TNFα is intended toinclude recombinant human TNFα (rhTNFα), which can be prepared bystandard recombinant expression methods or purchased commercially (R & DSystems, Catalog No. 210-TA, Minneapolis, Minn.). TNFα is also referredto as TNF.

The term “antibody”, as used herein, is intended to refer toimmunoglobulin molecules comprised of four polypeptide chains, two heavy(H) chains and two light (L) chains inter-connected by disulfide bonds.Each heavy chain is comprised of a heavy chain variable region(abbreviated herein as HCVR or VH) and a heavy chain constant region.The heavy chain constant region is comprised of three domains, CH1, CH2and CH3. Each light chain is comprised of a light chain variable region(abbreviated herein as LCVR or VL) and a light chain constant region.The light chain constant region is comprised of one domain, CL. The VHand VL regions can be further subdivided into regions ofhypervariability, termed complementarity determining regions (CDR),interspersed with regions that are more conserved, termed frameworkregions (FR). Each VH and VL is composed of three CDRs and four FRs,arranged from amino-terminus to carboxy-terminus in the following orderFR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The antibodies of the inventionare described in further detail in U.S. Pat. Nos. 6,090,382; 6,258,562;and 6,509,015, each of which is incorporated herein by reference in itsentirety. In one embodiment, the antibody of the invention is ananti-TNFα which interfere with TNFα activity. Examples of anti-TNFαantibodies include, hut are not limited to, anti-TNFα human antibodiesand antibody portions described herein as well as those described inU.S. Pat. Nos. 6,090,382; 6,258,562; 6,509,015, and in U.S. patentapplication Ser. Nos. 09/801,185 and 10/302,356, each of which isincorporated by reference herein. In one embodiment, the TNFα inhibitorused in the invention is an anti-TNFα antibody, or a fragment thereof,including infliximab (Remicade®, Johnson and Johnson; described in U.S.Pat. No. 5,656,272, incorporated by reference herein), CDP571 (ahumanized monoclonal anti-TNF-alpha IgG4 antibody), CDP 870 (a humanizedmonoclonal anti-TNF-alpha antibody fragment), an anti-TNF dAb (Peptech),CNTO 148 (golimumab; Medarex and Centocor), antibodies described in WO02/12502, and adalimumab (Humira® Abbott Laboratories, a human anti-TNFmAb, described in U.S. Pat. No. 6,090,382 as D2E7). Additional TNFantibodies which may be used in the invention are described in U.S. Pat.Nos. 6,593,458; 6,498,237; 6,451,983; and 6,448,380, each of which isincorporated by reference herein. The term includes the “antibody ofinterest” which is the antibody which is the target of the process ofthe invention.

The term “antigen-binding portion” of an antibody (or simply “antibodyportion”), as used herein, refers to one or more fragments of anantibody that retain the ability to specifically bind to an antigen(e.g., hTNFα). It has been shown that the antigen-binding function of anantibody can be performed by fragments of a full-length antibody.Examples of binding fragments encompassed within the term“antigen-binding portion” of an antibody include (i) a Fab fragment, amonovalent fragment consisting of the VL, VH, CL and CH1 domains; (ii) aF(ab′)₂ fragment, a bivalent fragment comprising two tab fragmentslinked by a disulfide bridge at the hinge region; (iii) a Fd fragmentconsisting of the VH and CH1 domains; (iv) a Fv fragment consisting ofthe VL and VH domains of a single arm of an antibody, (v) a dAb fragment(Ward et al. (1989) Nature 341:544-546), which consists of a VH domain;and (vi) an isolated complementarity determining region (CDR).Furthermore, although the two domains of the Fv fragment, VL and VH, arecoded for by separate genes, they can be joined, using recombinantmethods, by a synthetic linker that enables them to be made as a singleprotein chain in which the VL and VH regions pair to form monovalentmolecules (known as single chain Fv (scFv); see e.g., Bird et al. (1988)Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA85:5879-5883). Such single chain antibodies are also intended to beencompassed within the term “antigen-binding portion” of an antibody.Other forms of single chain antibodies, such as diabodies are alsoencompassed. Diabodies are bivalent, bispecific antibodies in which VHand VL domains are expressed on a single polypeptide chain, but using alinker that is too short to allow for pairing between the two domains onthe same chain, thereby forcing the domains to pair with complementarydomains of another chain and creating two antigen binding sites (seee.g., Holliger et al. (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448;Poljak et al. (1994) Structure 2:1121-1123). The antibody portions ofthe invention are described in further detail in U.S. Pat. Nos.6,090,382, 6,258,562, 6,509,015, each of which is incorporated herein byreference in its entirety.

Binding fragments are produced by recombinant DNA techniques, or byenzymatic or chemical cleavage of intact immunoglobulins. Bindingfragments include Fab, Fab′, F(ab′)₂, Fabc, Fv, single chains, andsingle-chain antibodies. Other than “bispecific” or “bifunctional”immunoglobulins or antibodies, an immunoglobulin or antibody isunderstood to have each of its binding sites identical. A “bispecific”or “bifunctional antibody” is an artificial hybrid antibody having twodifferent heavy/light chain pairs and two different binding sites.Bispecific antibodies can be produced by a variety of methods includingfusion of hybridomas or linking of Fab′ fragments. See, e.g.,Songsivilai & Lachmann, Clin. Exp. Immunol. 79:315-321 (1990); Kostelnyet al., J. Immunol. 148, 1547.1553 (1992).

A “conservative amino acid substitution”, as used herein, is one inwhich one amino acid residue is replaced with another amino acid residuehaving a similar side chain. Families of amino acid residues havingsimilar side chains have been defined in the art, including basic sidechains (e.g., lysine, arginine, histidine), acidic side chains (e.g.,aspartic acid, glutamic acid), uncharged polar side chains (e.g.,glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine),nonpolar side chains (e.g., alanine, valine, leucine, isoleucine,proline, phenylalanine, methionine, tryptophan), beta-branched sidechains (e.g., threonine, valine, isoleucine) and aromatic side chains(e.g., tyrosine, phenylalanine, tryptophan, histidine).

“Chimeric antibodies” refers to antibodies wherein one portion of eachof the amino acid sequences of heavy and light chains is homologous tocorresponding sequences in antibodies derived front a particular speciesor belonging to a particular class, while the remaining segment of thechains is homologous to corresponding sequences from another species. Inone embodiment, the invention features a chimeric antibody orantigen-binding fragment, in which the variable regions of both lightand heavy chains mimics the variable regions of antibodies derived fromone species of mammals, while the constant portions are homologous tothe sequences in antibodies derived from another species. In a preferredembodiment of the invention, chimeric antibodies are made by graftingCDRs from a mouse antibody onto the framework regions of a humanantibody.

“Humanized antibodies” refer to antibodies which comprise at least onechain comprising variable region framework residues substantially from ahuman antibody chain (referred to as the acceptor immunoglobulin orantibody) and at least one complementarity determining region (CDR)substantially from a non-human-antibody (e.g., mouse). In addition tothe grafting of the CDRs, humanized antibodies typically undergo furtheralterations in order to improve affinity and/or immunogenicity.

The term “multivalent antibody” refers to an antibody comprising morethan one antigen recognition site. For example, a “bivalent” antibodyhas two antigen recognition sites, whereas a “tetravalent” antibody hasfour antigen recognition sites. The terms “monospecific”, “bispecific”,“trispecific”, “tetraspecific”, etc. refer to the number of differentantigen recognition site specificities (as opposed to the number ofantigen recognition sites) present in a multivalent antibody. Forexample, a “monospecific” antibody's antigen recognition sites all bindthe same epitope. A “bispecific” or “dual specific” antibody has atleast one antigen recognition site that binds a first epitope and atleast one antigen recognition site that binds a second epitope that isdifferent from the first epitope. A “multivalent monospecific” antibodyhas multiple antigen recognition sites that all bind the same epitope. A“multivalent bispecific” antibody has multiple antigen recognitionsites, some number of which bind a first epitope and some number ofwhich bind a second epitope that is different from the first epitope

The term “human antibody”, as used herein, is intended to includeantibodies having variable and constant regions derived from humangermline immunoglobulin sequences. The human antibodies of the inventionmay include amino acid residues not encoded by human germlineimmunoglobulin sequences (e.g., mutations introduced by random orsite-specific mutagenesis in vitro or by somatic mutation in vivo), forexample in the CDRs and in particular CDR3. However, the term “humanantibody”, as used herein, is not intended to include antibodies inwhich CDR sequences derived from the germline of another mammalianspecies, such as a mouse, have been grafted onto human frameworksequences.

The term “recombinant human antibody”, as used herein, is intended toinclude all human antibodies that are prepared, expressed, created orisolated by recombinant means, such as antibodies expressed using arecombinant expression vector transfected into a host cell (describedfurther below), antibodies isolated from a recombinant, combinatorialhuman antibody library (described further below), antibodies isolatedfrom an animal (e.g., a mouse) that is transgenic for humanimmunoglobulin genes (see e.g., Taylor et al. (1992) Nucl. Acids Res.20:6287) or antibodies prepared, expressed, created or isolated by anyother means that involves splicing of human immunoglobulin genesequences to other DNA sequences. Such recombinant human antibodies havevariable and constant regions derived from human germline immunoglobulinsequences. In certain embodiments, however, such recombinant humanantibodies are subjected to in vitro mutagenesis (or, when an animaltransgenic for human Ig sequences is used, in vivo somatic mutagenesis)and thus the amino acid sequences of the VH and VL regions of therecombinant antibodies are sequences that, while derived from andrelated to human germline VH and VL sequences, may not naturally existwithin the human antibody germline repertoire in vivo.

Such chimeric, humanized, human, and dual specific antibodies can beproduced by recombinant DNA techniques known in the art, for exampleusing methods described in PCT International Application No.PCT/US86/02269; European Patent Application No. 184,187; European PatentApplication No. 171,496; European Patent Application No. 173,494; PCTinternational Publication No. WO 86/01533; U.S. Pat. No. 4,816,567;European Patent Application No. 125,023; Better et al. (1988) Science240:1041-1043; Liu et al. (1987) Proc. Natl. Acad. Sci. USA84:3439-3443; Liu et al. (1987) J. Immunol. 139:3521-3526; Sun et al.(1987) Proc. Natl. Acad. Sci. USA 84:214-218; Nishimura et al. (1987)Cancer Res. 47:999-1005; Wood et al. (1985) Nature 314:446-449; Shaw etal. (1988) J. Natl. Cancer Inst. 80:1553-1559); Morrison (1985) Science229:1202-1207; Oi et al. (1986) BioTechniques 4:214; U.S. Pat. No.5,225,539; Jones et al. (1986) Nature 321:552-525; Verhoeyan et al.(1988) Science 239:1534; and Beidler et al. (1988) J. Immunol.141:4053-4060, Queen et al., Proc. Natl. Acad. Sci. USA 86:10029-10033(1989), U.S. Pat. No. 5,530,101, U.S. Pat. No. 5,585,089, U.S. Pat. No.5,693,761, U.S. Pat. No. 5,693,762, Selick et al., WO 90/07861, andWinter, U.S. Pat. No. 5,225,539.

An “isolated antibody”, as used herein, is intended to refer to anantibody that is substantially free of other antibodies having differentantigenic specificities (e.g., an isolated antibody that specificallybinds hTNFα is substantially free of antibodies that specifically bindantigens other than hTNFα). An isolated antibody that specifically bindshTNFα may, however, have cross-reactivity to other antigens, such asTNFα molecules from other species (discussed in further detail below).Moreover, an isolated antibody may be substantially free of othercellular material and/or chemicals.

A “neutralizing antibody”, as used herein (or an “antibody thatneutralized hTNFα activity”), is intended to refer to an antibody whosebinding to hTNFα results in inhibition of the biological activity ofhTNFα. This inhibition of the biological activity of hTNFα can beassessed by measuring one or more indicators of hTNFα biologicalactivity, such as hTNFα-induced cytotoxicity (either in vitro or invivo), hTNFα-induced cellular activation and hTNFα binding to hTNFαreceptors. These indicators of hTNFα biological activity can be assessedby one or more of several standard in vitro or in vivo assays known inthe art (see U.S. Pat. No. 6,090,382). Preferably, the ability of anantibody to neutralize hTNFα activity is assessed by inhibition ofhTNFα-induced cytotoxicity of L929 cells. As an additional oralternative parameter of hTNFα activity, the ability of an antibody toinhibit hTNFα-induced expression of ELAM-1 on HUVEC, as a measure ofhTNFα-induced cellular activation, can be assessed.

The term “surface plasmon resonance”, as used herein, refers to anoptical phenomenon that allows for the analysis of real-time biospecificinteractions by detection of alterations in protein concentrationswithin a biosensor matrix, for example using the BIAcore system(Pharmacia Biosensor AB, Uppsala, Sweden and Piscataway, N.J.). Forfurther descriptions, see Example 1 of U.S. Pat. No. 6,258,562 andJönsson et al. (1993) Ann. Biol. Clin. 51:19; Jönsson et al. (1991)Biotechniques 11:620-627; Johnsson et al. (1995) J. Mol. Recognit.8:125; and Johnnson et al. (1991) Anal. Biochem. 198:268.

The term “K_(off)”, as used herein, is intended to refer to the off rateconstant for dissociation of an antibody from the antibody/antigencomplex.

The term “K_(d)”, as used herein, is intended to refer to thedissociation constant of a particular antibody-antigen interaction.

The term “IC₅₀” as used herein, is intended to refer to theconcentration of the inhibitor required to inhibit the biologicalendpoint of interest, e.g., neutralize cytotoxicity activity.

The term “nucleic acid molecule”, as used herein, is intended to includeDNA molecules and RNA molecules. A nucleic acid molecule may besingle-stranded or double-stranded, but preferably is double-strandedDNA.

The term “isolated nucleic acid molecule”, as used herein in referenceto nucleic acids encoding antibodies or antibody portions (e.g., VH, VL,CDR3) that bind hTNFα, is intended to refer to a nucleic acid moleculein which the nucleotide sequences encoding the antibody or antibodyportion are free of other nucleotide sequences encoding antibodies orantibody portions that bind antigens other than hTNFα, which othersequences may naturally flank the nucleic acid in human genomic DNA.Thus, for example, an isolated nucleic acid of the invention encoding aVH region of an anti-hTNFα antibody contains no other sequences encodingother Vi regions that bind antigens other than hTNFα.

The term “vector”, as used herein, is intended to refer to a nucleicacid molecule capable of transporting another nucleic acid to which ithas been linked. One type of vector is a “plasmid”, which refers to acircular double stranded DNA loop into which additional DNA segments maybe ligated. Another type of vector is a viral vector, wherein additionalDNA segments may be ligated into the viral genome. Certain vectors arecapable of autonomous replication in a host cell into which they areintroduced (e.g., bacterial vectors having a bacterial origin ofreplication and episomal mammalian vectors). Other vectors (e.g.,non-episomal mammalian vectors) can be integrated into the genome of ahost cell upon introduction into the host cell, and thereby arereplicated along with the host genome. Moreover, certain vectors arecapable of directing the expression of genes to which they areoperatively linked. Such vectors are referred to herein as “recombinantexpression vectors” (or simply, “expression vectors”). In general,expression vectors of utility in recombinant DNA techniques are often inthe form of plasmids. In the present specification. “plasmid” and“vector” may be used interchangeably as the plasmid is the most commonlyused form of vector. However, the invention is intended to include suchother forms of expression vectors, such as viral vectors (e.g.,replication defective retroviruses, adenoviruses and adeno-associatedviruses), which serve equivalent functions.

The term “recombinant host cell” (or simply “host cell”), as usedherein, is intended to refer to a cell into which a recombinantexpression vector has been introduced. It should be understood that suchterms are intended to refer not only to the particular subject cell butto the progeny of such a cell. Because certain modifications may occurin succeeding generations due to either mutation or environmentalinfluences, such progeny may not, in fact, be identical to the parentcell, but are still included within the scope of the term “host cell” asused herein.

The term “kit” as used herein refers to a packaged product or article ofmanufacture comprising components. The kit preferably comprises a box orcontainer that holds the components of the kit. The box or container isaffixed with a label or a Food and Drug Administration approvedprotocol. The box or container holds components of the invention whichare preferably contained within plastic, polyethylene, polypropylene,ethylene, or propylene vessels. The vessels can be capped-tubes orbottles. The kit can also include instructions for administering theTNFα antibody of the invention. In one embodiment the kit of theinvention includes the formulation comprising the human antibody D2E7,as described in PCT/IB03/04502 and U.S. application Ser. No. 10/222,140.

Various aspects of the invention are described in further detail herein.

II. Antibody Production

The invention herein provides methods for purifying an antibody from amixture comprising the antibody and one or more HCPs. The initialmixture is generally one resulting front the recombinant production ofthe antibody. Alternatively, the initial mixture may result fromproduction of the antibody by peptide synthesis (or other syntheticmeans) or the antibody may be purified from a native source of theantibody.

To express the antibodies, or antibody portions of the invention, DNAsencoding partial or full-length light and heavy chains are inserted intoexpression vectors such that the genes are operatively linked totranscriptional and translational control sequences. In this context,the term “operatively linked” is intended to mean that an antibody geneis ligated into a vector such that transcriptional and translationalcontrol sequences within the vector serve their intended function ofregulating the transcription and translation of the antibody gene. Theexpression vector and expression control sequences are chosen to becompatible with the expression host cell used. The antibody light chaingene and the antibody heavy chain gene can be inserted into separatevector or, more typically, both genes are inserted into the sameexpression vector. The antibody genes are inserted into the expressionvector by standard methods (e.g., ligation of complementary restrictionsites on the antibody gene fragment and vector, or blunt end ligation ifno restriction sites are present). Prior to insertion of the antibody orantibody-related light or heavy chain sequences, the expression vectormay already carry antibody constant region sequences. For example, oneapproach to converting the adalimumab or adalimumab-related VH and VLsequences to full-length antibody genes is to insert them intoexpression vectors already encoding heavy chain constant and light chainconstant regions, respectively, such that the VH segment is operativelylinked to the CH segment(s) within the vector and the VL segment isoperatively linked to the CL segment within the vector. Additionally oralternatively, the recombinant expression vector can encode a signalpeptide that facilitates secretion of the antibody chain front a hostcell. The antibody chain gene can be cloned into the vector such thatthe signal peptide is linked in-frame to the amino terminus of theantibody chain gene. The signal peptide can be an immunoglobulin signalpeptide or a heterologous signal peptide (i.e., a signal peptide from anon-immunoglobulin protein).

In addition to the antibody chain genes, the recombinant expressionvectors of the invention carry regulatory sequences that control theexpression of the antibody chain genes in a host cell. The term“regulatory sequence” is intended to include promoters, enhancers andother expression control elements (e.g., polyadenylation signals) thatcontrol the transcription or translation of the antibody chain genes.Such regulatory sequences are described, for example, in Goeddel: GeneExpression Technology; Methods in Enzymology 185, Academic Press, SanDiego, Calif. (1990). It will be appreciated by those skilled in the artthat the design of the expression vector, including the selection ofregulatory sequences may depend on such factors as the choice of thehost cell to be transformed, the level of expression of protein desired,etc. Preferred regulatory sequences for mammalian host cell expressioninclude viral elements that direct high levels of protein expression inmammalian cells, such as promoters and/or enhancers derived fromcytomegalovirus (CMV) (such as the CMV promoter/enhancer), Simian Virus40 (SV40) (such as the SV40 promoter/enhancer), adenovirus, (e.g., theadenovirus major late promoter (AdMLP)) and polyoma. For furtherdescription of viral regulatory elements, and sequences thereof, seee.g., U.S. Pat. No. 5,168,062 by Stinski, U.S. Pat. No. 4,510,245 byBell et al. and U.S. Pat. No. 4,968,615 by Schaffner et al.

In addition to the antibody chain genes and regulatory sequences, therecombinant expression vectors of the invention may carry additionalsequences, such as sequences that regulate replication of the vector inhost cells (e.g., origins of replication) and selectable marker genes.The selectable marker gene facilitates selection of host cells intowhich the vector has been introduced (sec e.g., U.S. Pat. Nos.4,399,216, 4,634,665 and 5,179,017, all by Axel et al.). For example,typically the selectable marker gene confers resistance to drugs, suchas G418, hygromycin or methotrexate, on a host cell into which thevector has been introduced. Preferred selectable marker genes includethe dihydrofolate reductase (DHFR) gene (for use in dhfr⁻ host cellswith methotrexate selection/amplification) and the neo gene (for G418selection).

For expression of the light and heavy chains, the expression vector(s)encoding the heavy and light chains is transfected into a host cell bystandard techniques. The various forms of the term “transfection” areintended to encompass a wide variety of techniques commonly used for theintroduction of exogenous DNA into a prokaryotic or eukaryotic hostcell, e.g., electroporation, calcium-phosphate precipitation,DEAE-dextran transfection and the like. Although it is theoreticallypossible to express the antibodies of the invention in eitherprokaryotic or eukaryotic host cells, expression of antibodies ineukaryotic cells, and most preferably mammalian host cells, is the mostpreferred because such eukaryotic cells, and in particular mammaliancells, are more likely than prokaryotic cells to assemble and secrete aproperly folded and immunologically active antibody. Prokaryoticexpression of antibody genes has been reported to be ineffective forproduction of high yields of active antibody (Boss and Wood (1985)Immunology Today 6:12-13).

Suitable host cells for cloning or expressing the DNA in the vectorsherein are the prokaryote, yeast, or higher eukaryote cells describedabove. Suitable prokaryotes for this purpose include eubacteria, such asGram-negative or Gram-positive organisms, for example,Enterobacteriaceae such as Escherichia, e.g., E. coli. Enterobacter,Erwinia, Klebsiella, Proteus, Salmonella, e.g., Salmonella typhimurium,Serratia, e.g., Serratia marcescans, and Shigella, as well as Bacillisuch as B. subtilis and B. licheniformis (e.g., B. licheniformis 41Pdisclosed in DD 266,710 published Apr. 12, 1989), Pseudomonas such as P.aeruginosa, and Streptomyces. One preferred E. coli cloning host is E.coli 294 (ATCC 31,446), although other strains such as E. coli B, E.coli X1776 (ATCC 31,537), and E. coli W3110 (ATCC 27,325) are suitable.These examples are illustrative rather than limiting,

In addition to prokaryotes, eukaryotic microbes such as filamentousfungi or yeast are suitable cloning or expression hosts for polypeptideencoding vectors. Saccharomyces, cerevisiae, or common baker's yeast, isthe most commonly used among lower eukaryotic host microorganisms.However, a number of other genera, species, and strains are commonlyavailable and useful herein, such as Schizosaccharomyces pombe;Kluyveromyces hosts such as, e.g., K. lactis, K. fragilis (ATCC 12,424),K. bulgaricus (ATCC 16,045), K. wickeramii (ATCC 24,178), K. waltii(ATCC 56,500), K. drosophilanum (ATCC 36,906), K. thermotolerans, and K.marxianus; yarrowia (EP 402,226); Pichia pastoris (EP 183,070); Candida;Trichodxerma reesia (EP 244,234); Neurospora crassa; Schwanniomyces suchas Schwanniomyces occidentalis; and filamentous fungi such as, e.g.,Neurospora, Penicillium, Tolypocladium, and Aspergillus hosts such as A.nidulans and A. niger.

Suitable host cells for the expression of glycosylated antibodies arederived from multicellular organisms. Examples of invertebrate cellsinclude plant and insect cells. Numerous baculoviral strains andvariants and corresponding permissive insect host cells from hosts suchas Spodoptera frugiperda (caterpillar), Aedes aegypti (mosquito, Aedesalbopictus (mosquito), Drosophila melanogaster (fruitfly), and Bombyxmori have been identified. A variety of viral strains for transfectionare publicly available, e.g., the L-1 variant of Autographa californicaNPV and the Bm-5 strain of Bombyx mori NPV, and such viruses may be usedas the virus herein according to the present invention, particularly fortransfection of Spodoptera frugiperda cells. Plant cell cultures ofcotton, corn, potato, soybean, petunia, tomato, and tobacco can also beutilized as hosts.

Preferred mammalian host cells for expressing the recombinant antibodiesof the invention include Chinese Hamster Ovary (CHO cells) (includingdhfr-CHO) cells, described in Urlaub and Chasin, (1980) PNAS USA77:4216-4220, used with a DHFR selectable marker, e.g., as described inKaufman and Sharp (1982) Mol. Biol. 1.59:601-621), NS0 myeloma cells,COS cells and SP2 cells. When recombinant expression vectors encodingantibody genes are introduced into mammalian host cells, the antibodiesare produced by culturing the host cells for a period of time sufficientto allow for expression of the antibody in the host cells or, morepreferably, secretion of the antibody into the culture medium in whichthe host cells are grown. Other examples of useful mammalian host celllines are monkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL1651); human embryonic kidney line (293 or 293 cells subcloned forgrowth in suspension culture, Graham et al., J. Gen Virol. 36:59(1977)); baby hamster kidney cells (BHK, ATCC CCL 10); Chinese hamsterovary cells/-DHFR (CHO, Urlaub et al., Proc. Natl. Acad. Sci. USA77:4216 (1980)); mouse sertoli cells (TM4, Mather, Biol. Reprod.23:243.251 (1980)) monkey kidney cells (CV1 ATCC CCL 70); African greenmonkey kidney ells (VERO-76, ATCC CRL-1587); human cervical carcinomacells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34);buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138,ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse mammary tumor(MMT 060562, ATCC CCL51); TRI cells (Mather et al., Annals N.Y. Acad.Sci. 383:44-68 (1982)); MRC 5 cells; FS4 cells; arld a human hepatomaline (Hep G2).

Host cells are transformed with the above-described expression orcloning vectors for antibody production and cultured in conventionalnutrient media modified as appropriate for inducing promoters, selectingtransformants, or amplifying the genes encoding the desired sequences.

The host cells used to produce an antibody may be cultured in a varietyof media. Commercially available media such as Ham's F10 (Sigma),Minimal Essential Medium ((MEM), (Sigma), RPMI-1640 (Sigma), andDulbecco's Modified Eagle's Medium ((DMEM), Sigma) are suitable forculturing the host cells. In addition, any of the media described in Hamet al., Meth. Enz. 58:44 (1979), Barnes et al., Anal. Biochem. 102:255(1980), U.S. Pat. Nos. 4,767,704; 4,657,866; 4,927,762; 4,560,655; or5,122,469; WO 90/03430; WO 87/00195; or U.S. Pat. No. Re. 30,985 may beused as culture media for the host cells. Any of these media may besupplemented as necessary with hormones and/or other growth factors(such as insulin, transferrin, or epidermal growth factor), salts (suchas sodium chloride, calcium, magnesium, and phosphate), buffers (such asHEPES), nucleotides (such as adenosine and thymidine), antibiotics (suchas gentamycin drug), trace elements (defined as inorganic compoundsusually present at final concentrations in the micromolar range), andglucose or an equivalent energy source. Any other necessary supplementsmay also be included at appropriate concentrations that would be knownto those skilled in the art. The culture conditions, such astemperature, pH, and the like, are those previously used with the hostcell selected for expression, and will be apparent to the ordinarilyskilled artisan.

Host cells can also be used to produce portions of intact antibodies,such as Fab fragments or scFv molecules. It is understood thatvariations on the above procedure are within the scope of the presentinvention. For example, it may be desirable to transfect a host cellwith DNA encoding either the light chain or the heavy chain (but notboth) of an antibody of this invention. Recombinant DNA technology mayalso be used to remove some or all of the DNA encoding either or both ofthe light and heavy chains that is not necessary for binding to hTNFα.The molecules expressed from such truncated DNA molecules are alsoencompassed by the antibodies of the invention. In addition,bifunctional antibodies may be produced in which one heavy and one lightchain are an antibody of the invention and the other heavy and lightchain are specific for an antigen other than hTNFα by crosslinking anantibody of the invention to a second antibody by standard chemicalcrosslinking methods.

In a preferred system for recombinant expression of an antibody, orantigen-binding portion thereof, of the invention, a recombinantexpression vector encoding both the antibody heavy chain and theantibody light chain is introduced into dhfr-CHO cells by calciumphosphate-mediated transfection Within the recombinant expressionvector, the antibody heavy and light chain genes are each operativelylinked to CMV enhancer/AdMLP promoter regulatory elements to drive highlevels of transcription of the genes. The recombinant expression vectoralso carries a DHFR gene, which allows for selection of CHO cells thathave been transfected with the vector using methotrexateselection/amplification. The selected transformant host cells areculture to allow for expression of the antibody heavy and light chainsand intact antibody is recovered from the culture medium. Standardmolecular biology techniques are used to prepare the recombinantexpression vector, transfect the host cells, select for transformants,culture the host cells and recover the antibody from the culture medium.

Recombinant human antibodies of the invention, including adalimumab oran antigen binding portion thereof, or adalimumab-related antibodiesdisclosed herein can be isolated by screening of a recombinantcombinatorial antibody library, preferably a scFv phage display library,prepared using human VL and VH cDNAs prepared from mRNA derived fronthuman lymphocytes. Methodologies for preparing and screening suchlibraries are known in the art. In addition to commercially availablekits for generating phage display libraries (e.g., the PharmaciaRecombinant Phage Antibody System, catalog no. 27-9400-01; and theStratagene SurfZAP™ phage display kit, catalog no. 240612), examples ofmethods and reagents particularly amenable for use in generating andscreening antibody display libraries can be found in, for example,Ladner et al. U.S. Pat. No. 5,223,409; Kang et al. PCT Publication No.WO 92/18619; Dower et al. PCT Publication No. WO 91/17271; Winter et al.PCT Publication No. WO 92/20791; Markland et al. PCT Publication No. WO92/15679; Breitling et a. PCT Publication No. WO 93/01288; McCafferty etal. PCT Publication No. WO 92/01047; Garrard et al. PCT Publication No.WO 92/09690; Fuchs et al. (1991) Bio/Technology 9:1370-1372; Hay et al.(1992) Hum Antibod Hybridomas 3:81-85; Huse et al. (1989) Science246:1275-1281; McCafferty et al., Nature (1990) 348:552-554; Griffithset al. (1993) EMBO J 12:725-734; Hawkins et al. (1992) J Mol Biol226:889-896; Clackson et al. (1991) Nature 352:624-628; Gram et al.(1992) PNAS 89:3576-3580; Garrard et al. (1991) Bio/Technology9:1373-1377; Hoogenboom et al. (1991) Nuc Acid Res 19:4133-4137; andBarbas et al. (1991) PNAS 88:7978-7982.

When using recombinant techniques, the antibody can be producedintracellularly, in the periplasmic space, or directly secreted into themedium. If the antibody is produced intracellularly, as a first step,the particulate debris, either host cells or lysed cells (e.g. resultingfrom homogenization), is removed, for example, by centrifugation orultrafiltration. Where the antibody is secreted into the medium,supernatant from such expression systems are generally firstconcentrated using a commercially available protein concentrationfilter, for example, an Amicon or Millipore Pellicon ultrafiltrationunit.

Prior to the process of the invention, procedures for purification fantibodies from cell debris initially depend on the site of expressionof the antibody. Some antibodies can be caused to be secreted directlyfrom the cell into the surrounding growth media; others are madeintracellularly. For the latter antibodies, the first step of apurification process involves: lysis of the cell, which can be done by avariety of methods, including mechanical shear, osmotic shock, orenzymatic treatments. Such disruption releases the entire contents ofthe cell into the homogenate, and in addition produces subcellularfragments that are difficult to remove due to their small size. Theseare generally removed by differential centrifugation or by filtration.Where the antibody is secreted it not be medium, supernatants from suchexpression systems are generally first concentrated using a commerciallyavailable protein concentration filter, for example, an Amicon orMillipore Pellicon ultrafiltration unit. Where the antibody is secretedinto the medium, the recombinant host cells can also be separated fromthe cell culture medium, for example, by tangential flow filtration.Antibodies can be further recovered from the culture medium using theantibody purification methods of the invention.

In one embodiment, the process of the invention includes humanantibodies, or antigen-binding portions thereof, that bind to human TNFαwith high affinity and a low off rate, and have a high neutralizingcapacity. Preferably, the human antibodies are recombinant, neutralizinghuman anti-hTNFα antibodies. The most preferred recombinant,neutralizing antibody used in the method of the invention is referred toherein us adalimumab, also referred to as adalimumab, Humira®, and D2E7(the amino acid sequence of the D2E7 VL region is shown in SEQ ID NO: 1;the amino acid sequence of the D2E7 VH region is shown in SEQ ID NO: 2).The properties of D2E7 (adalimumab; Humira®) have been described inSalfeld et al., U.S. Pat. Nos. 6,090,382, 6,258,562, and 6,509,015,which are each incorporated by reference herein. Other examples of TNFαantibodies include chimeric and humanized murine anti-hTNFα antibodieswhich have undergone clinical testing for treatment of rheumatoidarthritis (see e.g., Elliott et al. (1994) Lancet 344:1125-1127; Elliotet al, (1994) Lancet 344:1105-1110; Rankin et al. (1995) Br. J.Rheumatol. 34:334-342). In another embodiment, the TNFα antibody used inthe invention is infliximab (Remicade®, Johnson and Johnson; describedin U.S. Pat. No. 5,656,272, incorporated by reference herein), CDP571 (ahumanized monoclonal anti-TNF-alpha IgG4 antibody), CDP 870 (a humanizedmonoclonal anti-TNF-alpha antibody fragment), an anti-TNF dAb (Peptech),and CNTO 148 (golimumab; Medarex and Centocor, see also WO 02/12502).

In one embodiment, the methods of the invention include adalimumabantibodies and antibody portions, adalimumab-related antibodies andantibody portions, and other human antibodies and antibody portions withequivalent properties to adalimumab, such as high affinity binding tohTNFα with low dissociation kinetics and high neutralizing capacity. Inone embodiment, the invention provides treatment with an isolated humanantibody, or an antigen-binding portion there of, that dissociates fronthuman TNFα with a K_(d) of 1×10⁻⁸ M or less and a K_(off) rate constantof 1×10⁻³ s⁻¹ or less, both determined by surface plasmon resonance, andneutralizes human TNFα cytotoxicity in a standard in vitro L929 assaywith an IC₅₀ of 1×10⁻⁷ M or less. More preferably, the isolated humanantibody, or antigen-binding portion thereof, dissociates from humanTNFα with a K_(off) of 5×10⁻⁴ s⁻¹ or less, or even more preferably, witha K_(off) of 1×10⁻⁴ s⁻¹ or less. More preferably, the isolated humanantibody, or antigen-binding portion thereof, neutralizes human TNFαcytotoxicity in a standard in vitro L929 assay with an IC₅₀ of 1×10⁻⁸ Mor less, even more preferably with an IC₅₀ of 1×10⁻⁹ M or less and stillmore preferably with an IC₅₀ of 1×10⁻¹⁰ M or less. In a preferredembodiment, the antibody is an isolated human recombinant antibody, oran antigen-binding portion thereof.

It is well known in the art that antibody heavy and light chain CDR3domains play an important role in the binding specificity/affinity of anantibody for an antigen. Accordingly, in another aspect, the inventionpertains to methods of treating rheumatoid arthritis by administeringhuman antibodies obtained using the methods of the invention, whereinthe antibodies have slow dissociation kinetics for association withhTNFα and that have light and heavy chain CDR3 domains that structurallyare identical to or related to those of adalimumab. Position 9 of theadalimumab VL CDR3 can be occupied by Ala or Thr without substantiallyaffecting the K_(off). Accordingly, a consensus motif for the adalimumabVL CDR3 comprises the amino acid sequence: Q-R-Y-N-R-A-P-Y-(T/A) (SEQ IDNO: 3). Additionally, position 12 of the adalimumab VH CDR3 can beoccupied by Tyr or Asn, without substantially affecting the K_(off).Accordingly, a consensus motif for the adalimumab VH CDR3 comprises theamino acid sequence: V-S-Y-L-S-T-A-S-S-L-D-(Y/N) (SEQ ID NO: 4).Moreover, as demonstrated in Example 2 of U.S. Pat. No. 6,090,382, theCDR3 domain of the adalimumab heavy and light chains is amenable tosubstitution with a single alanine residue (at position 1, 4, 5, 7 or 8within the VL CDR3 or at position 2, 3, 4, 5, 6, 8, 9, 10 or 11 withinthe VH CDR3) without substantially affecting the K_(off). Still further,the skilled artisan will appreciate that, given the amenability of theadalimumab VL and VH CDR3 domains to substitutions by alanine,substitution of other amino acids within the CDR3 domains may bepossible while still retaining the low off rate constant of theantibody, in particular substitutions with conservative amino acids.Preferably, no more than one to five conservative amino acidsubstitutions are made within the adalimumab VL and/or VH CDR3 domains.More preferably, no more than one to three conservative amino acidsubstitutions are made within the adalimumab VL and/or VH CDR3 domains.Additionally, conservative amino acid substitutions should not be madeat amino acid positions critical for binding to hTNFα. Positions 2 and 5of the adalimumab VL CDR3 and positions 1 and 7 of the adalimumab VHCDR3 appear to be critical for interaction with hTNFα and thus,conservative amino acid substitutions preferably are not made at thesepositions (although an alanine substitution at position 5 of theadalimumab VL CDR3 is acceptable, as described above) (see U.S. Pat. No.6,090,382).

Accordingly, in another embodiment, the antibody or antigen-bindingportion thereof preferably contains the following characteristics:

a) dissociates from human TNFα with a K_(off) rate constant of 1×10⁻³s⁻¹ or less, as determined by surface plasmon resonance;

b) has a light chain CDR3 domain comprising the amino acid sequence ofSEQ ID NO: 3, or modified from SEQ ID NO: 3 by a single alaninesubstitution at position 1, 4, 5, 7 or 8 or by one to five conservativeamino acid substitutions at positions 1, 3, 4, 6, 7, 8 and/or 9:

c) has a heavy chain CDR3 domain comprising the amino acid sequence ofSEQ ID NO: 4, or modified from SEQ ID NO: 4 by a single alaninesubstitution at position 2, 3, 4, 5, 6, 8, 9, 10 or 11 or by one to fiveconservative amino acid substitutions at positions 2, 3, 4, 5, 6, 8, 9,10, 11 and/or 12.

More preferably, the antibody, or antigen-binding portion thereof,dissociates from human TNFα with a K_(off) of 5×10⁻⁴ s⁻¹ or less. Evenmore preferably, the antibody, or antigen-binding portion thereof,dissociates from human TNFα with a K_(off) of 1×10⁻⁴ s⁻¹ or less.

In yet another embodiment, the antibody or antigen-binding portionthereof preferably contains a light chain variable region (LCVR) havinga CDR3 domain comprising the amino acid sequence of SEQ ID NO: 3, ormodified from SEQ ID NO: 3 by a single alanine substitution at position1, 4, 5, 7 or 8, and with a heavy chain variable region (HCVR) having aCDR3 domain comprising the amino acid sequence of SEQ ID NO: 4, ormodified from SEQ ID NO: 4 by a single alanine substitution at position2, 3, 4, 5, 6, 8, 9, 10, or 11. Preferably, the LCVR further has a CDR2domain comprising the amino acid sequence of SEQ ID NO: 5 (i.e., theadalimumab VL CDR2) and the HCVR further has a CDR2 domain comprisingthe amino acid sequence of SEQ ID NO: 6 (i.e., the adalimumab VH CDR2).Even more preferably, the LCVR further has CDR1 domain comprising theamino acid sequence of SEQ ID NO: 7 (i.e., the adalimumab VL CDR1) andthe HCVR has a CDR1 domain comprising the amino acid sequence of SEQ IDNO: 8 (i.e., the adalimumab VH CDR1). The framework regions for VLpreferably are from the V_(K)I human germline family, more preferablyfront the A20 human germline Vk gene and most preferably from theadalimumab VL framework sequences shown in FIGS. 1A and 1B of U.S. Pat.No. 6,090,382. The framework regions for VH preferably are from theV_(H)3 human germline family, more preferably from the DP-31 humangermline VH gene and most preferably from the adalimumab VH frameworksequences shown in FIGS. 2A and 2B of U.S. Pat. No. 6,090,382.

Accordingly, in another embodiment, the antibody or antigen-bindingportion thereof preferably contains a light chain variable region (LCVR)comprising the amino acid sequence of SEQ ID NO: 1 (i.e., the adalimumabVL) and a heavy chain variable region (HCVR) comprising the amino acidsequence of SEQ ID NO: 2 (i.e., the adalimumab VH). In certainembodiments, the antibody comprises a heavy chain constant region, suchas an IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM or IgD constant region.Preferably, the heavy chain constant region is an IgG1 heavy chainconstant region or an IgG4 heavy chain constant region. Furthermore, theantibody can comprise a light chain constant region, either a kappalight chain constant region or a lambda light chain constant region.Preferably, the antibody comprises a kappa light chain constant region.Alternatively, the antibody portion can be, for example, a Fab fragmentor a single chain Fv fragment.

In still other embodiments, the antibody or antigen-binding portionthereof preferably contains adalimumab-related VL and VH CDR3 domains,for example, antibodies, or antigen-binding portions thereof, with alight chain variable region (LCVR) having a CDR3 domain comprising anamino acid sequence selected from the group consisting of SEQ ID NO: 3,SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14. SEQ ID NO:15, SEQ IT) NO: 16. SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19. SEQ IDNO: 20, SEQ ID NO: 21, SEQ ID NO: 22. SEQ ID NO: 23, SEQ ID NO: 24, SEQID NO: 25 and SEQ ID NO: 26 or with a heavy chain variable region (HCVR)having a CDR3 domain comprising an amino acid sequence selected from thegroup consisting of SEQ ID NO: 4, SEQ ID NO: 27, SEQ ID NO: 28, SEQ IDNO: 29, SEQ ID NO: 30, SEQ ID) NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQID NO: 34 and SEQ ID NO: 35.

The TNFα antibody used in the invention can be modified. In someembodiments, the TNFα antibody or antigen binding fragments thereof, ischemically modified to provide a desired effect. For example, pegylationof antibodies and antibody fragments of the invention may be carried outby any of the pegylation reactions known in the art, as described, forexample, in the following references: Focus on Growth Factors 3:4-10(1992); EP 0 154 316; and EP 0 401 384 (each of which is incorporated byreference herein in its entirety). Preferably, the pegylation is carriedout via an acylation reaction or an alkylation reaction with a reactivepolyethylene glycol molecule (or an analogous reactive water-solublepolymer). A preferred water-soluble polymer for pegylation of theantibodies and antibody fragments of the invention is polyethyleneglycol (PEG). As used herein, “polyethylene glycol” is meant toencompass any of the forms of PEG that have been used to derivativeother proteins, such as mono (Cl—ClO) alkoxy- or aryloxy-polyethyleneglycol.

Methods for preparing pegylated antibodies and antibody fragments of theinvention will generally comprise the steps of (a) reacting the antibodyor antibody fragment with polyethylene glycol, such as a reactive esteror aldehyde derivative of PEG, under conditions whereby the antibody orantibody fragment becomes attached to one or more PEG groups, and (b)obtaining the reaction products. It will be apparent to one of ordinaryskill in the art to select the optimal reaction conditions or theacylation reactions based on known parameters and the desired result.

Pegylated antibodies and antibody fragments may generally be used totreat TNFα-related disorders of the invention by administration of theTNFα antibodies and antibody fragments described herein. Generally thepegylated antibodies and antibody fragments have increased half-life, ascompared to the nonpegylated antibodies and antibody fragments. Thepegylated antibodies and antibody fragments may be employed alone,together, or in combination with other pharmaceutical compositions.

In yet another embodiment of the invention, TNFα antibodies or fragmentsthereof can be altered wherein the constant region of the antibody ismodified to reduce at least one constant region mediated biologicaleffector function relative to an unmodified antibody. To modify anantibody of the invention such that it exhibits reduced binding to theFc receptor, the immunoglobulin constant region segment of the antibodycan be mutated at particular regions necessary for Fe receptor (FcR)interactions (see e.g., Canfield and Morrison (1991) J. Exp. Med.173:1483-1491; and Lund et al. (1991) J. of Immunol. 147:2657-2662).Reduction in FcR binding ability of the antibody may also reduce othereffector functions which rely on FcR interactions, such as opsonizationand phagocytosis and antigen-dependent cellular cytotoxicity.

An antibody or antibody portion of the invention can be derivatized orlinked to another functional molecule (e.g., another peptide orprotein). Accordingly, the antibodies and antibody portions of theinvention are intended to include derivatized and otherwise modifiedforms of the human anti-hTNFα antibodies described herein, includingimmunoadhesin molecules. For example, an antibody or antibody portion ofthe invention can be functionally linked (by chemical coupling, geneticfusion, noncovalent association or otherwise) to one or more othermolecular entities, such as another antibody (e.g., a bispecificantibody or a diabody), a detectable agent, a cytotoxic agent, apharmaceutical agent, and/or a protein or peptide that can mediateassociate of the antibody or antibody portion with another molecule(such as a streptavidin core region or a polyhistidine tag).

One type of derivatized antibody is produced by crosslinking two or moreantibodies (of the same type or of different types, e.g., to createbispecific antibodies). Suitable crosslinkers include those that areheterobifunctional, having two distinctly reactive groups separated byan appropriate spacer (e.g., m-maleimidobenzoyl-N-hydroxysuccinimideester) or homobifunctional (e.g., disuccinimidyl suberate). Such linkersare available from Pierce Chemical Company, Rockford, Ill.

Useful detectable agents with which an antibody or antibody portion ofthe invention may be derivatized include fluorescent compounds.Exemplary fluorescent detectable agents include fluorescein, fluoresceinisothiocyanate, rhodamine, 5-dimethylamine-1-napthalenesulfonylchloride, phycoerythrin and the like. An antibody may also bederivatized with detectable enzymes, such as alkaline phosphatase,horseradish peroxidase, glucose oxidase and the like. When an antibodyis derivatized with a detectable enzyme, it is detected by addingadditional reagents that the enzyme uses to produce a detectablereaction product. For example, when the detectable agent horseradishperoxidase is present, the addition of hydrogen peroxide anddiaminobenzidine leads to a colored reaction product, which isdetectable. An antibody may also be derivatized with biotin, anddetected through indirect measurement of avidin or streptavidin binding.

An antibody, or antibody portion, of the invention can be prepared byrecombinant expression of immunoglobulin light and heavy chain genes ina host cell. To express an antibody recombinantly, a host cell istransfected with one or more recombinant expression vectors carrying DNAfragments encoding the immunoglobulin light and heavy chains of theantibody such that the light and heavy chains are expressed in the hostcell and, preferably, secreted into the medium in which the host cellsare cultured, from which medium the antibodies can be recovered.Standard recombinant DNA methodologies are used to obtain antibody heavyand light chain genes, incorporate these genes into recombinantexpression vectors and introduce the vectors into host cells, such asthose described in Sambrook, Fritsch and Maniatis (eds), MolecularCloning; A Laboratory Manual, Second Edition, Cold Spring Harbor, N.Y.,(1989), Ausubel et al. (eds.) Current Protocols in Molecular Biology,Greene Publishing Associates, (1989) and in U.S. Pat. No. 4,816,397 byBoss et al.

To express adalimumab or a adalimumab-related antibody, DNA fragmentsencoding the light and heavy chain variable regions are first obtained.These DNAs can be obtained by amplification and modification of germlinelight and heavy chain variable sequences using the polymerase chainreaction (PCR). Germline DNA sequences for human heavy and light chainvariable region genes are known in the art (see e.g., the “Vbase” humangermline sequence database; see also Kabat et al. (1991) Sequences atProteins of Immunological Interest, Filth Edition, U.S. Department ofHealth and Human Services, NIH Publication No. 91-3242; Tomlinson et al.(1992) “The Repertoire of Human Germline V_(H) Sequences Reveals aboutFifty Groups of V_(H) Segments with Different Hypervariable Loops” J.Mol. Bol. 227:776-798; and Cox et al. (1994) “A Directory of HumanGerm-line V₇₈ Segments Reveals a Strong Bias in their Usage” Eur. J.Immunol. 24:827-836 the contents of each of which are expresslyincorporated herein by reference). To obtain a DNA fragment encoding theheavy chain variable region of adalimumab, or a adalimumab-relatedantibody, a member of the V_(H)3 family of human germline VH genes isamplified by standard PCR. Most preferably, the DP-31 VH germlinesequence is amplified. To obtain a DNA fragment encoding the light chainvariable region of adalimumab, or a adalimumab-related antibody, amember of the V_(K)I family of human germline VL genes is amplified bystandard PCR. Most preferably, the A20 VL germline sequence isamplified. PCR primers suitable for use in amplifying the DP-31 germlineVH and A20 germline VL sequences can be designed based on the nucleotidesequences disclosed in the references cited supra, using standardmethods.

Once the germline VH and VL fragments are obtained, these sequences canbe mutated to encode the adalimumab or adalimumab-related amino acidsequences disclosed herein. The amino acid sequences encoded by thegermline VH and VL DNA sequences are first compared to the adalimumab oradalimumab-related VH and VL amino acid sequences to identify amino acidresidues in the adalimumab or adalimumab-related sequence that differfrom germline. Then, the appropriate nucleotides of the germline DNAsequences are mutated such that the mutated germline sequence encodesthe adalimumab or adalimumab-related amino acid sequence, using thegenetic code to determine which nucleotide changes should be made.Mutagenesis of the germline sequences is carried out by standardmethods, such as PCR-mediated mutagenesis (in which the mutatednucleotides are incorporated into the PCR primers such that the PCRproduct contains the mutations) or site-directed mutagenesis.

Once DNA fragments encoding adalimumab or adalimumab-related VH and VLsegments are obtained (by amplification and mutagenesis of germline VHand VL genes, as described above), these DNA fragments can be furthermanipulated by standard recombinant DNA techniques, for example toconvert the variable region genes to full-length antibody chain genes,to Fab fragment genes or to a scFv gene. In these manipulations, a VL-or VH-encoding DNA fragment is operatively linked to another DNAfragment encoding another protein, such as an antibody constant regionor a flexible linker. The term “operatively linked”, as used in thiscontext, is intended to mean that the two DNA fragments are joined suchthat the amino acid sequences encoded by the two DNA fragments remainin-frame.

The isolated DNA encoding the VH region can be converted to, alull-length heavy chain gene by operatively linking the VH-encoding DNAto another DNA molecule encoding heavy chain constant regions (CH1, CH2and CH3). The sequences of human heavy chain constant region genes areknown in the an (see e.g., Kabat et al. (1991) Sequence of Proteins ofImmunological Interest, Fifth Edition, U.S. Department of Health andHuman Services, NIH Publication No. 91-3242) and DNA fragmentsencompassing these regions can be obtained by standard PCRamplification. The heavy chain constant region can be an IgG1, IgG2,IgG3, IgG3, IgA, IgE, IgM or IgD constant region, but most preferably isan IgG1 or IgG4 constant region. For a Fab fragment heavy chain gene,the VH-encoding DNA can be operatively linked to another DNA moleculeencoding only the heavy chain CH1 constant region.

The isolated DNA encoding the VL region can be converted to afull-length light chain gene (as well as a Fab light chain gene) byoperatively linking the VL-encoding DNA to another DNA molecule encodingthe light chain constant region, CL. The sequences of human light chainconstant region genes are known in the art (see e.g., Kabat et al.(1991) Sequences of Proteins of Immunological Interest, Fitih Edition,U.S. Department of Health and Human Services, NIH Publication No.91-3242) and DNA fragments encompassing these regions can be obtained bystandard PCR amplification. The light chain constant region can be akappa or lambda constant region, but most preferably is a kappa constantregion.

To create a scFv gene, the VH- and VL-encoding DNA fragments areoperatively linked to another fragment encoding a flexible linker, e.g.,encoding the amino acid sequence (Gly₄-Ser)₃, such that the VH and VLsequences can be expressed as a contiguous single-chain protein, withthe VL and VH regions joined by the flexible linker (see e.g., Bird etal. (1988) Science 242:423-426; Huston et al. (1988) Proc. Natl. Acad.Sci. USA 85:5879-5883; McCafferty et al., Nature (1990) 3348:552-554).

To express the antibodies, or antibody portions of the invention, DNAsencoding partial or full-length light and heavy chains, obtained asdescribed above, are inserted into expression vectors such that thegenes are operatively linked to transcriptional and translationalcontrol sequences. In this context, the term “operatively linked” isintended to mean that an antibody gene is ligated into a vector suchthat transcriptional and translational control sequences within thevector serve their intended function of regulating the transcription andtranslation of the antibody gene. The expression vector and expressioncontrol sequences are chosen to be compatible with the expression hostcell used. The antibody light chain gene and the antibody heavy chaingene can be inserted into separate vector or, more typically, both genesare inserted into the same expression vector. The antibody genes areinserted into the expression vector by standard methods (e.g., ligationof complementary restriction sites on the antibody gene fragment andvector, or blunt end ligation if no restriction sites are present).Prior to insertion of the adalimumab or adalimumab-related light orheavy chain sequences, the expression vector may already carry antibodyconstant region sequences. For example, one approach to converting theadalimumab or adalimumab-related VH and VL sequences to full-lengthantibody genes is to insert them into expression vectors alreadyencoding heavy chain constant and light chain constant regions,respectively, such that the VH segment is operatively linked to the CHsegment(s) within the vector and the VL segment is operatively linked tothe CL segment within the vector. Additionally or alternatively, therecombinant expression vector can encode a signal peptide thatfacilitates secretion of the antibody chain from a host cell. Theantibody chain gene can be cloned into the vector such that the signalpeptide is linked in-frame to the amino terminus of the antibody chaingene. The signal peptide can be an immunoglobulin signal peptide or aheterologous signal peptide (i.e., a signal peptide from anon-immunoglobulin protein).

In addition to the antibody chain genes, the recombinant expressionvectors of the invention carry regulatory sequences that control theexpression of the antibody chain genes in a host cell. The term“regulatory sequence” is intended to include promoters, enhancers andother expression control elements (e.g., polyadenylation signals) thatcontrol the transcription or translation of the antibody chain genes.Such regulatory sequences are described, for example, in Goeddel; GeneExpression Technology: Methods in Enzymology 185, Academic Press, SanDiego. Calif. (1990). It will be appreciated by those skilled in the artthat the design of the expression vector, including the selection ofregulatory sequences may depend on such factors as the choice of thehost cell to be transformed, the level of expression of protein desired,etc. Preferred regulatory sequences for mammalian host cell expressioninclude viral elements that direct high levels of protein expression inmammalian cells, such as promoters and/or enhancers derived fromcytomegalovirus (CMV) (such as the CMV promoter/enhancer), Simian Virus40) (SV40) (such as the SV40 promoter/enhancer), adenovirus, (e.g., theadenovirus major late promoter (AdMLP)) and polyoma. For furtherdescription of viral regulatory elements, and sequences thereof, seee.g., U.S. Pat. No. 5,168,062 by Stinski, U.S. Pat. No. 4,510,245 byBell et al. and U.S. Pat. No. 4,968,615 by Schaffner et al.

In addition to the antibody chain genes and regulatory sequences, therecombinant expression vectors of the invention may carry additionalsequences, such as sequences that regulate replication of the vector inhost cells (e.g., origins of replication) and selectable marker genes.The selectable marker gene facilitates selection of host cells intowhich the vector has been introduced (see e.g., U.S. Pat. Nos.4,399,216, 4,634,665 and 5,179,017, all by Axel et al.). For example,typically the selectable marker gene confers resistance to drugs, suchas G418, hygromycin or methotrexate, on a host cell into, which thevector has been introduced. Preferred selectable marker genes includethe dihydrofolate reductase (DHFR) gene (for use in dhfr-host cells withmethotrexate selection/amplification) and the neo gene (for G418selection).

For expression of the light and heavy chains, the expression vector(s)encoding the heavy and light chains is transfected into a host cell bystandard techniques. The various forms of the term “transfection” areintended to encompass a wide variety of techniques commonly used for theintroduction of exogenous DNA into a prokaryotic or eukaryotic hostcell, e.g., electroporation, calcium-phosphate precipitation,DEAE-dextran transfection and the like. Although it is theoreticallypossible to express the antibodies of the invention in eitherprokaryotic or eukaryotic host cells, expression of antibodies ineukaryotic cells, and most preferably mammalian host cells, is the mostpreferred because such eukaryotic cells, and in particular mammaliancells, are more likely than prokaryotic cells to assemble and secrete aproperly folded and immunologically active antibody. Prokaryoticexpression of antibody genes has been reported to be ineffective forproduction of high yields of active antibody (Boss and Wxood (1985)Immunology Today 6:12-13).

Preferred mammalian host cells for expressing the recombinant antibodiesof the invention include Chinese Hamster Ovary (CHO cells) (includingdhfr− CHO cells, described in Urlaub and Chasin, (1980) PNAS USA77:4216-4220, used with a DHFR selectable marker, e.g., as described inKaufman and Sharp (1982) Mol. Biol. 159:601-621), NS0 myeloma cells, COScells and SP2 cells. When recombinant expression vectors encodingantibody genes are introduced into mammalian host cells, the antibodiesare produced by culturing the host cells for a period of time sufficientto allow for expression of the antibody in the host cells or, morepreferably, secretion of the antibody into the culture medium in whichthe host cells are grown. Antibodies can be recovered from the culturemedium using protein purification methods.

Host cells can also be used to produce portions of intact antibodies,such as Fab fragments or scFv molecules. It is understood thatvariations on the above procedure are within the scope of the presentinvention. For example, it may be desirable to transfect a host cellwith DNA encoding either the light chain or the heavy chain (but notboth) of an antibody of this invention. Recombinant DNA technology mayalso be used to remove some or all of the DNA encoding either or both ofthe light and heavy chains that is not necessary for binding to hTNFα.The molecules expressed from such truncated DNA molecules are alsoencompassed by the antibodies of the invention. In addition,bifunctional antibodies may be produced in which one heavy and one lightchain are an antibody of the invention and the other heavy and lightchain are specific for an antigen other than hTNFα by crosslinking anantibody of the invention to a second antibody by standard chemicalcrosslinking methods.

In a preferred system for recombinant expression of an antibody, orantigen-binding portion thereof, of the invention, a recombinantexpression vector encoding both the antibody heavy chain and theantibody light chain is introduced into dhfr-CHO cells by calciumphosphate-mediated transfection. Within the recombinant expressionvector, the antibody heavy and light chain genes are each operativelylinked to CMV enhancer/AdMLP promoter regulatory elements to drive highlevels of transcription of the genes. The recombinant expression vectoralso carries a DHFR gene, which allows for selection of CHO cells thathave been transfected with the vector using methotrexateselection/amplification. The selected transformant host cells areculture to allow for expression of the antibody heavy and light chainsand intact antibody is recovered from the culture medium. Standardmolecular biology techniques are used to prepare the recombinantexpression vector, transfect the host cells, select for transformants,culture the host cells and rtecover the antibody from the culturemedium.

Recombinant human antibodies of the invention in addition to adalimumabor an antigen binding portion thereof, or adalimumab-related antibodiesdisclosed herein can be isolated by screening of a recombinantcombinatorial antibody library, preferably a scFv phage display library,prepared using human VL and VH cDNAs prepared from mRNA derived fromhuman lymphocytes. Methodologies for preparing and screening suchlibraries are known in the art. In addition to commercially availablekits for generating phage display libraries (e.g., the PharmaciaRecombinant Phage Antibody System, catalog no. 27-9400-01; and theStratagene SurfZAP™ phage display kit, catalog no. 240612), examples ofmethods and reagents particularly amenable for use in generating andscreening antibody display libraries can be found in, for example,Ladner et al. U.S. Pat. No. 5,223,409; Kang et al. PCT Publication No.WO 92/18619; Dower et al. PC Publication No. WO 91/17271; Winter et al.PCT Publication No. WO 92/20791; Markland et al. PCT Publication No. WO92/15679; Breitling et al. PCT Publication No. WO 93/01288; McCaffertyet al. PCT Publication No. WO 92/01047; Garrard et al. PCT PublicationNo. WO 92/09690; Fuchs et al. (1991) Bio/Technology 9: 1370-1372; Hay etal. (1992) Hum Antibod Hybridomas 3:81-85; Huse et al. (1989) Science246:1275-1281; McCafferty et al., Nature (1990) 348:552-554; Griffithset al. (1993) EMBO J 12:725-734; Hawkins et al. (1992) J Mol Biol226:889-896; Clackson et al. (1991) Nature 352:624-628; Gram et al.(1992) PNAS 9:3576-3580; Garrard et al. (1991) Bio/Technology9:1373-1377; Hoogenboom et al. (1991) Nuc Acid Res 19:4133-4137; andBarbas et al. (1991) PNAS 88:7978-7982.

In a preferred embodiment, to isolate human antibodies with highaffinity and a low off rate constant for hTNFα, a murine anti-hTNFαantibody having high affinity and a low off rate constant for hTNFα(e.g., MAK 195, the hybridoma for which has deposit number ECACC 87050801) is first used to select human heavy and light chain sequenceshaving similar binding activity toward hTNFα, using the epitopeimprinting methods described in Hoogenboom et al., PCT Publication No.WO 93/06213. The antibody libraries used in this method are preferablyscFv libraries prepared and screened as described in McCafferty et al.,PCT Publication No. WO 92/01047, McCafferty et al. Nature (1990)348:552-554; and Griffiths et al. (1993) EMBO J 12:725-734. The scFvantibody libraries preferably are screened using recombinant human TNFαas the antigen.

Once initial human VL and VH segments are selected, “mix and match”experiments, in which different pairs of the initially selected VL andVH segments are screened for hTNFα binding, are performed to selectpreferred VL/VH pair combinations. Additionally, to further improve theaffinity and/or lower the off rate constant for hTNFα binding, the VLand VH segments of the preferred VL/VH pair(s) can be randomly mutated,preferably within the CDR3 region of VH and/or VL, in a processanalogous to the in vive somatic mutation process responsible foraffinity maturation of antibodies during a natural immune response. Thisin vitro affinity maturation can be accomplished by amplifying VH and VLregions using PCR primers complimentary to the VH CDR3 or VL CDR3,respectively, which primers have been “spiked” with a random mixture ofthe four nucleotide bases at certain positions such that the resultantPCR products encode VH and VL segments into which random mutations havebeen introduced into the VH and/or VL CDR3 regions. These randomlymutated VH and VL segments can be rescreened for binding to hTNFα, andsequences that exhibit high affinity and a low off rate for hTNFαbinding can be selected.

Following screening and isolation of an anti-hTNFα antibody of theinvention from a recombinant immunoglobulin display library, nucleicacid encoding the selected antibody can be recovered from the displaypackage (e.g., from the phage genome) and subcloned into otherexpression vectors by standard recombinant DNA techniques. If desired,the nucleic acid can be further manipulated to create other antibodyforms of the invention (e.g., linked to nucleic acid encoding additionalimmunoglobulin domains, such as additional constant regions). To expressa recombinant human antibody isolated by screening of a combinatoriallibrary, the DNA encoding the antibody is cloned into a recombinantexpression vector and introduced into a mammalian host cells, asdescribed in further detail in above.

Methods of isolating human antibodies with high affinity and a low offrate constant for hTNFα are also described in U.S. Pat. Nos. 6,090,382,6,258,562, and 6,509,015, each of which is incorporated by referenceherein.

III. Antibody Purification

The invention provides an method for producing an HCP-reduced antibodypreparation from a mixture comprising an antibody and at least one HCP.The invention also provides a method for producing a procathepsinL-reduced antibody preparation from a mixture comprising an antibody andat least one procathepsin L. The purification process of the inventionbegins at the separation step when the antibody has been produced usingmethods described in Section II and conventional methods in the art.Typically in the art, antibody-HCP mixtures are subjected to protein Acapture (e.g., a protein A column) as an initial purification step,since the antibody hinds to protein A whereas HCP will flow through. Thepurification methods of the invention have the advantage that it is notnecessary to subject the mixture comprising an antibody and at least oneHCP to protein A capture (e.g., a protein A column) as an initial step,or as any step in the purification method.

Once a clarified solution or mixture comprising the antibody has beenobtained, separation of the antibody from the other proteins produced bythe cell, such as HCPs, is performed using a combination of differentpurification techniques, including ion exchange separation step(s) andhydrophobic interaction separation step(s). The separation stepsseparate mixtures of proteins on the basis of their charge, degree ofhydrophobicity, and/or size. In one embodiment of the invention,separation is performed using chromatography, including cationic,anionic, and hydrophobic. Several different chromatography resins areavailable for each of these techniques, allowing accurate tailoring ofthe purification scheme to the particular protein involved. The essenceof each of the separation methods is that proteins can be caused eitherto move at different rates down a long column, achieving a physicalseparation that increases as they pass further down the column, or toadhere selectively to the separation medium, being then differentiallyeluted by different solvents. In some cases, the antibody is separatedfront impurities when the impurities specifically adhere to the column,and the antibody does not, that is, the antibody is present in theflowthrough.

Methods of purifying antibodies from undesired proteins are providedbelow, e.g., process A. In one embodiment, the invention includes thesteps, individually or in combination, described below in Process A.Process A provides a method of purifying a mixture comprising anantibody using ion exchange separation (cation exchange chromatographyand anion exchange chromatography) and hydrophobic interactionseparation, resulting in an antibody preparation suitable for use in apharmaceutical composition. Process A has the advantage that it can becarried out without the need to perform protein A capture as an initialstep in antibody purification. In one embodiment, the antibody purifiedusing process A is adalimumab. Process A generally comprises thefollowing:

A mixture comprising an antibody and impurities, e.g., HCP(s) is loadedonto an ion exchange column, such as a cation exchange column. Themixture may be loaded at a load of about ≦30 g antibody/L per cycle. Themixture loaded onto the cation column is subsequently washed with washbuffer (equilibration buffer). The antibody is then eluted from thecolumn, and a first eluate is obtained.

The first eluate is then often virally inactivated and pH adjusted inpreparation for anion exchange chromatography. The first eluate isvirally inactivated by adjusting the pH to a low pH relative to theelution buffer (described further in section IIIC). The pH of thevirally inactivated eluate is subsequently adjusted in more than onestep to a final pH of about 7.6, which is the pH of the anion exchangecolumn which follows in sequence.

Following viral inactivation, the first eluate is often subjected to asecond ion exchange separation step, where the first eluate is loadedonto an anion exchange column (e.g., a Q Sepharose column). The columnis washed with a wash buffer, and a first flowthrough comprising theantibody is obtained.

The flowthrough is further purified by loading it onto a hydrophobicinteraction column (phenyl sepharose). The column is washed, and theantibody is eluted from the column such that a second eluate isobtained.

Process B is described below and provides an improved method forproducing a host cell protein-(HCP) reduced antibody preparation from amixture comprising an antibody. The language “reduced” when referring toHCP or procathepsin L, includes improvements over levels, e.g.,concentration or activity, of HCP or procathepsin L at comparable pointsin process A. In one embodiment, the first eluate of process B comprisesa reduced level of HCP or procathepsin L in comparison to the firsteluate of process A. In one embodiment, the first flowthrough of processB comprises a reduced level of HCP or procathepsin L in comparison tothe first flowthrough of process A. In one embodiment, the second eluateof process B comprises a reduced level of HCP or procathepsin L incomparison to the second eluate of process A. In another embodiment, theantibody preparation resulting from process B comprises a reduced levelof HCP or procathepsin L in comparison the antibody preparationresulting from process A.

Process B Generally Comprises the Following:

A mixture comprising an antibody and impurities, e.g., HCP(s), is loadedonto an ion exchange column, such as a cation exchange column. Themixture may be loaded at a load of about ≦35 g antibody/L per cycle atpH 7 or at a load of about ≦70 g antibody/L per cycle at pH 5. Themixture loaded onto the cation column is subsequently washed with washbuffer (equilibration buffer). Following the equilibration wash buffer,an intermediate wash step is performed, wherein the column is washedwith an intermediate buffer which has similar conductivity to theelution buffer. This intermediate wash step improves clearance ofprocess-related impurities. The antibody is then eluted from the columnusing elution buffer, and a first eluate is obtained.

The first eluate is then virally inactivated and pH adjusted inpreparation for anion exchange chromatography. The first eluate isvirally inactivated by adjusting the pH to a low pH relative to theelution buffer. The pH and conductivity of the virally inactivatedeluate is subsequently adjusted in one step to a final pH of about7.8-8.2, which is the pH of the equilibrated anion exchange column whichfollows in sequence.

Following viral inactivation, the first eluate is subjected to a secondion exchange separation step, where the first eluate is loaded onto ananion exchange column (e.g., a Q Sepharose column). The column is washedwith a wash buffer, and a first flowthrough comprising the antibody isobtained.

The flowthrough is further purified by loading it onto a hydrophobicinteraction column (phenyl sepharose). The column is washed, and theantibody is eluted from the column such that a second eluate isobtained. The result of process B is a preparation having reduced HCPs,including procathepsin L. Further results of process B include theremoval of process bottlenecks, e.g., higher productivity created incell culture scale-up, by moving HCP clearance to the front part of theprocess and an overall improvement in antibody yield. Additional detailsregarding the improved process of the invention are provided below.

III.A. Ion Exchange Separation

The present invention features methods for producing a HCP-reducedantibody preparation from a mixture comprising an antibody and at leastone HCP by subjecting the mixture to at least one ion exchangeseparation step such that a first eluate comprising the antibody isobtained. Ion exchange separation includes any method by which twosubstances are separated based on the difference in their respectiveionic charges.

In performing the separation, the antibody mixture may be contacted withthe ion exchange material, e.g., using a batch purification technique orchromatography. For example, for batch purification, ion exchangematerial is prepared in or equilibrated to the desired starting buffer.A slurry of the ion exchange material is obtained. The antibody solutionis contacted with the slurry to adsorb the antibody to be separated tothe ion exchange material. The solution comprising the HCP(s) that donot bind to the ion exchange material is separated from the slurry,e.g., by allowing the slurry to settle and removing the supernatant. Theslurry can be subjected to one or more wash steps. If desired, theslurry can be contacted with a solution of higher conductivity to desorbHCPs that have bound to the ion exchange material. In order to elutebound polypeptides, the salt concentration may be increased.

Ion exchange chromatography may also be used as an ion exchangeseparation technique. Ion exchange chromatography separates moleculesbased on differences between the overall charge of the molecules. Forthe purification of an antibody, the antibody must have a chargeopposite to that of the functional group attached to the ion exchangematerial, e.g., resin, in order to bind. For example, antibodies, whichgenerally have an overall positive charge in the buffer pH below its pI,will bind well to cation exchange material, which contain negativelycharged functional groups.

In ion exchange chromatography, charged patches on the surface of thesolute are attracted by opposite charges attached to a chromatographymatrix, provided the ionic strength of the surrounding buffer is low.Elution is generally achieved by increasing the ionic strength (i.e.,conductivity; of the buffer to compete with the solute for the chargedsites of the ion exchange matrix. Changing the pH and thereby alteringthe charge of the solute is another way to achieve elution of thesolute. The change in conductivity or pH may be gradual (gradientelution) or stepwise (step elution).

Anionic or cationic substituents may be attached to matrices in order toform anionic or cationic supports for chromatography. Anionic exchangesubstituents include diethylaminoethyl(DEAE), quaternary aminoethyl(QAE)and quaternary amine (Q) groups. Cationic substitutents includecarboxymethyl (CM), sulfoethyl(SE), sulfopropyl(SP), phosphate (P) andsulfonate(S). Cellulose ion exchange resins such as DE23, DE32, DE52,CM-23, CM-32 and CM-52 are available from Whatnian Ltd. Maidstone, Kent,U.K. SEPHADEX®-based and -locross-linked ion exchangers are also known.For example, DEAE-, QAE-, CM-, and SP-SEPHADEX® and DEAE-, Q-, CM- andS-SEPHAROSE® and SEPHAROSE® Fast Flow are all available from PharmaciaAB. Further, both DEAE and CM derivatized ethylene glycol-methacrylatecopolymer such as TOYOPEARL DEAE-650S or M and TOYOPEARL CM-650S or Mare available from Toso Haas Co., Philadelphia, Pa.

In one embodiment of the invention, the mixture comprising an antibodyand at least one HCP is loaded onto a cation exchange (CEX)chromatography column. The mixture is loaded onto the CEX column in aloading buffer which may be the same as the equilibration buffer used toequilibrate the column. As the HCP-comprising mixture passes over theCEX column, the target protein is adsorbed to the CEX resin and variousHCPs (such as host cell proteins, where the target protein is producedin a recombinant host cell, or other process-derived impurities)flowthrough or bind weakly or nonspecifically to the CEX resin. Invarious embodiments, the CEX resin is a synthetic methacrylate basedpolymeric resin attached to a sulfonate group (Fractogel SO₃. (FractogelS)). In one embodiment, the equilibration buffer comprises 20 mM Na₂PO₄,25 mM NaCl, pH 6.8. Other suitable equilibration buffers include, forexample, BIS and HEPES at physiological concentrations, for example,concentrations in the range between about 0.5 mM and about 100 mM (e.g.,10 mM, 20 mM, 50 mM, etc.), and physiological salt concentrations (e.g.,about 0.15 mM NaCl), and at pH from 5.0-9.0.

In exemplary embodiments, the CEX chromatography is a Fractogel Scolumn. In one embodiment, about 30 gram (g) antibody per liter (L)resin to about 40 g antibody per L resin is loaded onto the Fractogel Scolumn. In another embodiment, about 35 g antibody per L resin is loadedonto the Fractogel S column at pH 7. It has been discovered that aloading amount of about 35 g antibody per L resin at pH 7 increases theclearance of impurities, e.g., HCP(s) and procathepsin L. The acceptableoperating ranges of a Fractogel S column to be used in the method of theinvention are described in Table 1 below.

TABLE 1 Acceptable operating ranges for Fractogel S chromatography at pH7 Operating parameter AOR Resin capacity ≦35 g protein/L resin Loadsample pH 6.5-7.5 Effective load dilution 1:1-1:2 Wash 2 elution buffer1:3-1:4 concentrate to WFI ratio Linear velocity 75-300 cm/hr

It further has been discovered that the loading capacity of the columncan be increased by carrying out the chromatography at pH 5. Inparticular, it has been discovered that a loading amount of about 70 gantibody per L resin at pH 5 increases the clearance of impurities,e.g., HCP(s) and procathepsin L. Accordingly, in a pH range of about pH5 to about pH 7, a loading amount of about 35 g to about 70 g antibodyper L of resin can be used.

Following the loading of the antibody mixture onto the column, the CEXresin is then washed with a wash buffer. In particular, it has beendiscovered that a plurality of wash steps with different wash buffersresults in a further HCP-reduced antibody preparation. Specifically, ithas been discovered that procathepsin L levels can be reduced by the useof a first wash step and an intermediate wash step using a wash bufferand an intermediate wash buffer, respectively. In one embodiment, theCEX resin is first washed with a wash buffer which is the same as theequilibration buffer. In certain embodiments, the wash buffer is 20 mMNa₂PO₄, 25 mM NaCl, pH 6.8. Other suitable wash buffers include, forexample, HIS and HEPES at physiological concentrations, for example,concentration in the range between about 0.5 mM and about 100) mM (e.g.,10 mM, 20 mM, 50 mM, etc.), and physiological salt concentrations (e.g.,about 0.15 mM NaCl), and at pH from 5.0-9.0.

In a preferred embodiment of the invention, an intermediate wash step isperformed. It has been discovered that reduced levels of HCP in general,and in particular, procathepsin L can be achieved by using anintermediate wash buffer comprising, in part, the same buffer as the CEXelution buffer. The improved reduction of HCP in general, and inparticular procathepsin L, results in part, from the increasedconductivity in the intermediate wash buffer. Increasing theconductivity of the intermediate wash buffer causes charge displacementof the HCP(s), which has a lower pI relative to the that of theantibody, thus causing the weaker binding HCP(s) to wash through thecolumn. Increased clearance of the weaker binding impurities, e.g.,HCP(s) including procathepsin L, in turn provides more binding area forthe target substance, e.g., the antibody. In other embodiments, theintermediate wash buffer contains from about 40% to about 50% elationbuffer and from about 50% to about 60% water for injection. In furtherembodiments, the intermediate wash buffer contains 45% elution bufferand 55% water for injection. In one embodiment, the wash buffer used inthe intermediate wash is 20 mM Na₂PO₄, 150 mM sodium chloride, pH 7.

Following a plurality of washes, the antibody is eluted from the firstcationic exchange material such that a first eluate having a reducedlevel of HCP is obtained. The first eluate also has a reduced level ofprocathepsin L in view of the intermediate wash step. In one embodiment,the first eluate obtained using the method of the invention comprises anabout 3 to an about 5 fold decrease in HCP levels in comparison to acomparable step of process A. In another embodiment, the first eluateobtained using the method of the invention comprises an about 2 to anabout 3 fold decrease in cathepsin L activity in comparison to acomparable step of process A. In one embodiment, the first eluatecomprises a range of about 90 to about 100 fold less HCP than themixture as determined by a HCP ELISA. In another embodiment, the firsteluate comprises cathepsin L activity ranging from between about 25 toabout 60 RFU/s/mg of antibody as measured by a cathepsin L kinetic assay

In a preferred embodiment, the initial eluate comprising the antibody ispassed over a second IE material and a flowthrough comprising a furtherreduced level of HCP is obtained. In some embodiments, the second IEstep may be batch purification as described infra. In other embodiments,the second IF step comprises loading the first eluate onto a second ionexchange chromatography column, washing the column and obtaining a firstflowthrough. The second IE material may be anion exchange (AEX) resin,e.g., Q sepharose column. In some embodiments, between about 30 gantibody per L resin and about 40 g antibody per L or resin is loadedonto the anion exchange column. Increasing the loading amount betweenabout 40 g antibody per L resin and about 50 g antibody per L resincauses a decrease in clearance of impurities, e.g., HCP(s). As theHCP-comprising mixture passes over the AEX column, the various HCP(s)bind to the AEX resin, and the antibody passes through or bindsnonspecifically to the AEX resin. In certain embodiments, the anionexchange resin is Q Sepharose.

Often, the antibody mixture to be purified will be present in a bufferfrom the previous purification step. Many buffers are available and canbe selected by routine experimentation. For example, an equilibrationbuffer of 25 mM trolamine, 40 mM NaCl, pH 8 may be used. In oneembodiment, prior to passing the initial eluate over the second IEmaterial, the second IE material may be equilibrated with equilibrationbuffer. This may be done, for example, by altering the pH andconductivity of the first eluate such that the pH and conductivity ofthe first eluate is substantially similar or corresponds to the pH andconductivity of the equilibrated second IE material, i.e., altering thepH and conductivity of the first eluate such that it corresponds to thatof the equilibrated second ion exchange material. In some embodiments,the pH of the AEX material (e.g. Q Sepharose) is adjusted withequilibration buffer to range from about 7.7 to about 8.3. In furtherembodiments, the pH of the CEX eluate (e.g., initial eluate) is adjustedto range about 7.7 to about 8.3. In certain embodiments, the pH of boththe AEX material and the initial eluate is about 8. In some embodiments,the conductivity of the AEX material ranges from about 3.5 mS/cm toabout 4.9 mS/cm. In further embodiments, the conductivity of the initialeluate ranges from about 3.5 mS/cm to about 4.9 mS/cm. It has beendiscovered that adjustment of the load conductivity and pH to that ofthe conductivity and pH of the second ion exchange material enhancesimpurity clearance. In relation to process A, it has been discoveredthat an overall decrease in conductivity and/or an increase in pH of thefirst eluate and/or equilibrated second ion exchange material results inincreased HCP(s) clearance.

Following the loading of the antibody mixture onto the column, the AEXresin is then washed with a wash buffer. The wash buffer may be the sameas the equilibration buffer, e.g., 25 mM trolamine, 40 mM NaCl, pH 8. Inone embodiment, the wash may be pooled with the flowthrough such that afirst flowthrough comprising the antibody and having a reduced level ofHCP is obtained. In further embodiments, the first flowthrough has areduced level of procathepsin L. In one embodiment, the firstflowthrough obtained using the method of the invention comprises anabout 7 to an about 700 told decrease in HCP levels in comparison to acomparable step of process A. In another embodiment, the firstflowthrough obtained using the method of the invention comprises anabout 6 to an about 25 fold decrease in cathepsin L activity incomparison to a comparable step of process A. In other embodiments, thefirst flowthrough comprises a range of about 840 to about 850 fold lessHCP than the first eluate as determined by a HCP ELISA. In yet anotherembodiment, the first flowthrough comprises cathepsin L activity rangingfrom between about 0.4 to about 4 RFU/s/mg of antibody as measured by acathepsin L kinetic assay

Acceptable operating ranges for Q sepharose chromatography to be used inthe method of the invention are described below in Table 2:

TABLE 2 Acceptable operating ranges for Q Sepharose FF chromatographyParameter AOR Load conductivity 4.0-5.5 mS/cm Load pH 7.8-8.2 Columnloading ≦40 g/L Linear velocity 150-300 cm/hr

The use of a cationic exchange material versus an anionic exchangematerial is based on the overall charge of the protein as discussedsupra. Therefore, it is within the scope of this invention to employ ananionic exchange material prior to the use of a cationic exchangematerial. Furthermore, it is within the scope of this invention toemploy only a cationic exchange material or only an anionic exchangematerial.

The methods of the present invention can optionally include furtherpurification steps. Examples of additional purification procedures whichmay be performed prior to, during, or following the ion exchangechromatography method include fractionation on a hydrophobic interactionchromatography (e.g. on phenyl sepharose), ethanol precipitation,isoelectric focusing, Reverse Phase HPLC, chromatography on silica,chromatography on heparin sepharose, further anion exchangechromatography and/or further cation exchange chromatography,chromatofocusing. SDS-PAGE, ammonium sulfate precipitation,hydroxylapatite chromatography, gel electrophoresis, dialysis, andaffinity chromatography (e.g. using protein A, protein G, an antibody, aspecific substrate, ligand or antigen as the capture reagent).

III.B. Hydrophobic Interaction Separation

The present invention also features methods for producing a HCP-reducedantibody preparation from a mixture comprising an antibody and at leastone HCP further comprising a hydrophobic interaction separation stepwherein the first flowthrough is subjected to a first hydrophobicinteraction material such that a second eluate having a reduced level ofHCP is obtained.

In performing the separation, the polypeptide mixture may be contactedwith the HIC material, e.g., using a batch purification technique orusing a column. Prior to HIC purification it may be desirable to removeany chaotropic agents or very hydrophobic substances, e.g., by passingthe mixture through a precolumn.

For example, for batch purification, HIC material is prepared in orequilibrated to the desired equilibration buffer. A slurry of the HICmaterial is obtained. The antibody solution is contacted with the slurryto adsorb the antibody to be separated to the HIC material. The solutioncomprising the HCPs that do not bind to the HIC material is separatedfrom the slurry, e.g., by allowing the slurry to settle and removing thesupernatant. The slurry can be subjected to one or more washing steps.If desired, the slurry can be contacted with a solution of lowerconductivity to desorb antibodies that have bound to the HIC material.In order to elute bound antibodies, the salt concentration can bedecreased.

In other embodiments, the hydrophobic interaction separation stepcomprises loading the first flowthrough onto a column comprising a firsthydrophobic interaction material and washing the first hydrophobicinteraction material such that a second eluate is obtained.

The hydrophobic interaction separation step may include a hydrophobicinteraction chromatography (HIC) step. Whereas ion exchangechromatography relies on the charges of proteins, e.g., antibodies, toisolate them, hydrophobic interaction chromatography uses thehydrophobic properties of some proteins, e.g., antibodies. Hydrophobicgroups on the antibody bind to hydrophillic groups on the column. Themore hydrophobic a protein is, the stronger it will bind to the column.The HIC step removes, for example, host cell derived impurities (e.g.,DNA and other high and low molecular weight product-related species).

Hydrophobic interactions are strongest at high ionic strength,therefore, this form of separation is conveniently performed followingsalt precipitations or ion exchange procedures. Adsorption of theantibody to a HIC column is favored by high salt concentrations, but theactual concentrations can vary over a wide range depending on the natureof the antibody and the particular HIC ligand chosen. Various ions canbe arranged in a so-called soluphobic series depending on whether theypromote hydrophobic interactions (salting-out effects) or disrupt thestructure of water (chaotropic effect) and lead to the weakening of thehydrophobic interaction. Cations are ranked in terms of increasingsalting out effect as Ba⁺⁺<; Ca⁺⁺<; Mg⁺⁺<; Li⁺<; Cs⁺<; Na⁺<; K⁺<; Rb⁺<;NH₄ ⁺, while anions may be ranked in terms of increasing chaotropiceffect as PO⁻<; SO₄ ⁻<; CH₃COOO⁻<; Cl⁻<; Br⁻<; NO₃ ⁻<; ClO₄ ⁻<; I⁻<;SCN⁻

In general, Na, K or NH₄ sulfates effectively promote ligand-proteininteraction in HIC. Salts may be formulated that influence the strengthof the interaction as given by the following relationship: (NH₄)₂SO₄>;Na₂SO₄>; NaCl>; NH₄Cl>; NaBr>; NaSCN. In general, salt concentrations ofbetween about 0.75 and about 2M ammonium sulfate or between about 1 and4M NaCl are useful.

HIC columns normally comprise a base matrix (e.g. cross-linked agaroseor synthetic copolymer material) to which hydrobobic ligands (e.g. alkylor aryl groups) are coupled. The preferred HIC column comprises anagarose resin substituted with phenyl groups (e.g. a Phenyl Sepharose™column). Many IIIC columns are available commercially. Examples include,but are not limited to, Phenyl Sepharose™ 6 Fast Flow column with low orhigh substitution (Pharmacia LKB Biotechnology, AB, Sweden); PhenylSepharose™ High Performance column (Pharmacia LKB Biotechnology, AB.Sweden); Octyl Sepharose™ High Performance column (Pharmacia LKBBiotechnology, AB, Sweden); Fractogel™ EMD Propyl or Fractogel™ EMDPhenyl columns (E. Merck, Germany); Macro-Prep™ Mehyl or Macro-Prep™t-Butyl Supports (Bio-Rad, California); WP III-Propyl (C.sub.3)™ column(J. T. Baker, New Jersey); and Toyopearl™ ether, phenyl or butyl columns(TosoHaas, Pa.).

Following any preliminary purification step(s), the mixture comprisingthe antibody of interest and HCP(s) may be subjected to HIC. Often, theantibody composition to be purified will be present in a buffer from theprevious purification step. However, it may be necessary to add a bufferto the antibody composition prior to the HIC step. Many buffers areavailable and can be selected by routine experimentation. In oneembodiment, the pH of the mixture comprising the antibody to be purifiedand at least one HCP in a loading buffer is adjusted to a pH of about 7using either an acid or base, depending on the starting pH, and aconductivity of about 136 to about 158 mS/cm. In one embodiment, theantibody mixture is diluted with a buffer comprising 40 mM sodiumphosphate, 2.2 M (NH₄)₂SO₄, pH 7.

Prior to loading the antibody mixture, the column may be equilibratedwith equilibration buffer. In some embodiments, the equilibration bufferis 20 mM sodium phosphate, 1.1 M (NH₄)₂SO₄, pH 7.

In one embodiment, the mixture, e.g., first flowthrough comprising theantibody, is loaded onto a phenyl sepharose HIC column. In certainembodiments, the protein loading for this step ranges between about 20and about 40 g protein per L of resin. In other embodiments, the proteinloading for this step is about 35 g protein per L of resin. In someembodiments, two or three chromatography cycles may be required toprocess the entire quantity of available material.

Following binding of the protein to the hydrophobic interaction column,the column may be washed with a wash buffer that may be the same as theequilibration buffer, e.g., 1.1 M (NH₄)₂SO₄, pH 7.

The antibody is eluted from the column using an elution buffer such thata second eluate is obtained. The elution buffer can be selected usingroutine experimentation. The pH of the elution buffer ranges betweenabout 6 and about 8 and has a low ammonium sulfate concentration (i.e.,less than about 1 M (NH₄)₃SO₄). The conductivity of the elution bufferranges from about 87 to about 101 mS/cm. In one embodiment, the elutionbuffer contains 11 mM sodium phosphate, 0.625 M (NH₄)₂SO₄), pH 7. It hasbeen discovered that lower salt concentrations result in less adalimumabbinding to the resin. The antibody is eluted from the second ionexchange material such that a second eluate having a reduced level ofHCP is obtained. The second eluate also has a reduced level ofprocathepsin L. In one embodiment, the second obtained using the methodof the invention comprises an about 10 to an about 96 fold decrease inHCP levels in comparison to a comparable step of process A. In anotherembodiment, the second eluate obtained using the method of the inventioncomprises an about 5 to an about 15 fold decrease in cathepsin Lactivity in comparison to a comparable step of process A. In oneembodiment, the second eluate comprises a range of about 3 to about 5fold less HCP than the first flowthrough as determined by a HCP ELISA.In another embodiment, the second eluate comprises cathepsin L activityranging from between about 0.5 to about 1.5 RFU/s/mg of antibody asmeasured by a cathepsin L kinetic assay.

Acceptable operating ranges for the phenyl sepharose chromatographycolumn used in the methods of the invention are shown below in Table 3.

TABLE 3 Acceptable operating ranges for Phenyl Sepharose HPchromatography Operating parameter AOR Column loading 20-40 g/L Loadsample dilution 0.9:1 to 1.1:1 Linear velocity 25-125 cm/hr

Further purification steps can include virus removing steps as well asnanofiltration, ultrafiltration and/or diafiltration steps, as describedherein.

III.C. Viral Inactivation

In order to provide a margin of safety, potential undetected viruses areinactivated during the purification process. Methods of viralinactivation are known in the art and include heat inactivation(pasteurization), pH inactivation, solvent/detergent treatment, UV andgamma ray irradiation and the addition of certain chemical inactivatingagents such as β-propiolactone or e.g. copper phenanthroline as in U.S.Pat. No. 4,534,972, etc. In some embodiments, subjecting the mixture toviral inactivation can include pH viral inactivation. Methods of pHviral inactivation techniques are also well known in the art. Forinstance, typical methods of viral inactivation include incubating themixture for a period of time at low pH, subsequently neutralizing the pHand removing particulates by filtration. The choice of pH level largelydepends on the stability profile of the antibody product and buffercomponents. It is known that the quality of the target antibody duringlow pH virus inactivation is affected by pH and the duration of the lowpH incubation. Virus inactivation is dependent on these same parametersin addition to protein concentration, which may reduce inactivation athigh concentrations. Thus, the proper parameters of proteinconcentration, pH and duration of inactivation may be selected byroutine experimentation.

The pH of the mixture may be lowered by any suitable acid including, butnot limited to, citric acid, acetic acid, caprylic acid, or othersuitable acids. In preferred embodiments, the pH of the mixture isadjusted with 1 M citric acid.

In some embodiments, the mixture is incubated at pH from about 2.9 toabout 3.9 for about 15 minutes to about 180 minutes. In furtherembodiments, the mixture is incubated at about pH 3.5 for about 60minutes to about 120 minutes. In still further embodiments, the mixtureis incubated at about pH 3.5 for about 60 minutes to about minutes.

In one embodiment, the mixture comprising the antibody and HCPs issubjected to viral inactivation prior to IE separation. In otherembodiments, the initial eluate is subjected to viral inactivation priorto IE separation. In certain embodiments, the initial eluate issubjected to viral inactivation prior to anion exchange chromatography.

Following viral inactivation, the mixture is adjusted as needed forfurther purification steps. For example, the pH-adjusted pool may besubjected to filtration. In one embodiment, following low pH viralinactivation and/or filtration, the pH of the mixture is typicallyadjusted to a more neutral pH, e.g., from about 6.5 to about 8.5. Forexample, the mixture may be flushed with water for injection (WFI) toobtain the desired pH.

The low pH virus inactivation parameters used in the method of theinvention are shown in Table 4 below.

TABLE 4 Acceptable operating parameters for low pH virus inactivationOperating parameter AOR Incubation pH 3.0-3.7 Incubation time 60-180 minProtein concentration ≦33 g/L

The invention includes a method where the first eluate from the ionexchange column is subjected to viral inactivation prior to the secondion exchange chromatography step. In one embodiment, viral inactivationis achieved through pH viral inactivation.

IV. Method for Determining Host Cell Protein (HCP) Levels

The present invention also provides methods for determining the residuallevels of Host Cell Protein (HCP) concentration in the purified antibodycomposition. As described above, HCPs are desirably excluded from thefinal target substance product, e.g., the antibody. Exemplary HCPsinclude proteins originating from the source of the antibody production.Failure to identify and sufficiently remove HCPs from the targetantibody may lead to reduced efficacy and/or adverse patient reactions.

As used herein, the term “HCP ELISA” refers to an ELISA where the secondantibody used in the assay is specific to the HCPs produced from cells,e.g., CHO cells, used to generate the antibody, e.g., adalimumab. Thesecond antibody may be produced according to conventional methods knownto those of skill in the art. For example, the second antibody may beproduced using HCPs obtained by sham production and purification runs.i.e., the same cell line used to produce the antibody of interest isused, but the cell line is not transfected with antibody DNA. In anexemplary embodiment, the second antibody is produced using HPCs similarto those expressed in the cell expression system of choice. i.e., thecell expression system used to produce the target antibody.

Generally, HCP ELISA comprises sandwiching a liquid sample comprisingHCPs between two layers of antibodies, i.e., a first antibody and asecond antibody. The sample is incubated during which time the HCPs inthe sample are captured by the first antibody, e.g., goat anti-CHO,affinity purified (Cygnus). A labeled second antibody specific to theHCPs produced from the cells used to generate the antibody, e.g.,anti-CHO HCP Biotinylated, is added, and binds to the HCPs within thesample. The amount of HCP contained in the sample is determined usingthe appropriate test based on the label of the second antibody.

HCP ELISA may be used for determining the level of HCPs in an antibodycomposition, such as an eluate or flowthrough obtained using the processdescribed in section III above. The present invention also provides acomposition comprising an antibody, wherein the composition has nodetectable level of HCPs as determined by an HCP Enzyme LinkedImmunosorbent Assay (“ELISA”). In one embodiment, the first eluatecomprises between about 12,000 to about 19,500 ng/mg of HCP. In oneembodiment, the second eluate comprises between about 1.0 and about 0.0ng/mg of HCP.

V. Method for Determining Procathepsin L Levels

The invention provides a kinetic assay (or cathepsin L kinetic assay)for determining the amount of procathepsin L in a sample. Procathepsin Lis a host cell protein derived from certain expression systems and, uponactivation to cathepsin L, is known to cause fragmentation of proteins,including antibodies such as adalimumab. Studies have demonstrated thatprocathepsin L is synthesized as an inactive zymogen and later processedto the active cathepsin L form. Activation of procathepsin L occurs byproteolytic removal of the N-terminal pro-peptide region by either otherproteases such as cathepsin D or by autocatalytic activation within theacidic conditions of the lysosome (Turk et al. (1999) Eur J Biochem259:929). Furthermore, Mason et al. (see Mason et al. (1992) BiochemBiophysical Res Comm 189: 1659) report the activation of cathepsin L canbe achieved to a higher degree at pH 5.5 with the addition of negativelycharged molecules, such as dextran sulfate, at lower pH conditions.

Previous methods of detecting levels of procathepsin L (or the activeform cathepsin L) included analytical methods such as weak anionexchange chromatography. Such methods are limited, however, when testingin-process samples. i.e., samples obtained from the process describedabove in section III, due to buffer system interference and matrixeffects. Thus, the invention provides a high throughput fluorescentenzymatic method to better monitor procathepsin L, for example, for thepurpose of process monitoring.

The kinetic assay of the invention provides a method of determiningprocathepsin L at levels which cannot be readily detected by standardend point assays. The kinetic assay also provides a means of determiningwhether the level of procathepsin L is reproducibly low. In oneembodiment, samples may be obtained from any point in the processdescribed in Section III, in order to confirm or determine that thelevel of procathepsin L is being reduced in the overall process.Procathepsin L is activated by removing the amino terminal from theprotein. In one embodiment, activation is achieved using a peptidase,such as, but not limited to, cathepsin D. Once activated, cathepsin Lcan selectively hydrolyze substrates. A substrate is contacted with thesample and monitored for cathepsin L activity based on changes to thesubstrate.

In a preferred embodiment, the substrate for cathepsin L comprises alabel. The label may include any agent which allows the cathepsinactivity to be determined. Examples of labeled substrates whichcathepsin L can selectively hydrolyze include synthetic substrates suchas Z-leucine-arginine-AMC (R & D Systems). The peptide substrate maycontain a fluorescent 7-amino-4-methyl coumarin (AMC) group that isquenched by the amide bond between the amino group of the AMC and thecarboxyl group of the arginine. Upon cleavage of the amide bond bycathepsin L, the released AMC group is fluorescent and can be measuredby excitation and emission wavelengths of 380 nm and 40 nm respectively.This excitation may be measured and used to determine the level ofcathepsin L activity. The rate of substrate turnover is directlyproportional to the amount of cathepsin L present in the sample. Thismeasurement is used in combination with a reference sample having knowncathepsin L activity and known amount of cathepsin L. The cathepsin Lactivity in the sample is then correlated to the amount of antibodypresent in the sample. In one embodiment, the first eluate comprisescathepsin L activity ranging from between about 25 to about 60 RFU/s/mgantibody. In another embodiment, the first flowthrough comprisescathepsin L activity ranging from between about 0.4 to about 4 RFU/s/mgantibody. In one embodiment, the second eluate comprises cathepsin Lactivity ranging from between about 0.5 to about 1.5 RFU/s/ing antibody.

In one embodiment, the kinetic assay comprises determining the amount ofprocathepsin L in a material derived from a mammalian cell expressionsystem comprising by contacting the material with an enzyme to processprocathepsin L to the active cathepsin L form, such that a cathepsin Lsample is obtained. Once activated, cathepsin L can selectivelyhydrolyze substrates, including synthetic substrates such asZ-leucine-arginine-AMC. A substrate is then added to the sample,including, for example Z-leucine-arginine-AMC, which contains afluorescent 7-amino-4-methyl coumarin (AMC) group that is quenched bythe amide bond between the amino group of the AMC and the carboxyl groupof the arginine. Upon cleavage of the amide bond by cathepsin L, thereleased AMC group is fluorescent and can be measured by excitation andemission wavelengths of 380 nm and 460 nm respectively. The determinedcathepsin L activity is used as an indication of the amount ofprocathepsin L in the material derived front the mammalian cellexpression system, e.g., Chinese Hamster Ovary (CHO) cells.

In one embodiment, the first eluate comprises cathepsin L activityranging from between about 25 to about 60 RFU/s/mg antibody. In anotherembodiment, the first flowthrough comprises cathepsin L activity rangingfrom between about 0.4 to about 4 RFU/s/ng antibody. In one embodiment,the second eluate comprises cathepsin L activity ranging from betweenabout 0.5 to about 1.5 RFU/s/mg antibody.

The invention also encompasses ranges intermediate to the above recitedamounts are also intended to be part of this invention. For example,ranges of values using a combination of any of the above recited valuesa upper and/or lower limits are intended to be included, as well as anynumber between the described range.

The invention includes any of the above-mentioned modifications, aloneor in combination with one another.

V. Pharmaceutical Compositions

Antibodies obtained using the process of the invention may beincorporated into pharmaceutical compositions suitable foradministration to a subject. Typically, the pharmaceutical compositioncomprises an antibody, or antigen-binding portion there f, and apharmaceutically acceptable carrier. As used herein, “pharmaceuticallyacceptable carrier” includes any and all solvents, dispersion media,coatings, antibacterial and antifungal agents, isotonic and absorptiondelaying agents, and the like that are physiologically compatible.Examples of pharmaceutically acceptable carriers include one or more ofwater, saline, phosphate buffered saline, dextrose, glycerol, ethanoland the like, as well as combinations thereof. In many cases, it ispreferable to include isotonic agents, for example, sugars, polyalcoholssuch as mannitol, sorbitol, or sodium chloride in the composition.Pharmaceutically acceptable carriers may further comprise minor amountsof auxiliary substances such as wetting or emulsifying agents,preservatives or buffers, which enhance the shelf life or effectivenessof antibody, or antigen-binding portion thereof.

Pharmaceutical compositions comprising antibodies, or antigen-bindingportions thereof, purified using the methods of the invention may befound in a variety of forms. These include, for example, liquid,semi-solid and solid dosage forms, such as liquid solutions (e.g.,injectable and infusible solutions), dispersions or suspensions,tablets, pills, powders, liposomes and suppositories. The preferred formdepends on the intended mode of administration and therapeuticapplication. Typical preferred compositions are in the form ofinjectable or infusible solutions, such as compositions similar to thoseused for passive immunization of humans with other antibodies or otherTNFα inhibitors. The preferred mode of administration is parenteral(e.g., intravenous, subcutaneous, intraperitoneal, intramuscular). In apreferred embodiment, the antibody is administered by intravenousinfusion or injection. In another preferred embodiment, the antibody isadministered by intramuscular or subcutaneous injection.

Therapeutic compositions typically must be sterile and stable under theconditions of manufacture and storage. The composition can be formulatedas a solution, microemulsion, dispersion, liposome, or other orderedstructure suitable to high drug concentration. Sterile injectablesolutions can be prepared by incorporating the active compound (i.e.,antibody, or antigen-binding portion thereof) in the required amount inan appropriate solvent with one or a combination of ingredientsenumerated above, as required, followed by filtered sterilization.Generally, dispersions are prepared by incorporating the active compoundinto a sterile vehicle that contains a basic dispersion medium and therequired other ingredients from those enumerated above. In the case ofsterile powders for the preparation of sterile injectable solutions, thepreferred methods of preparation are vacuum drying and freeze-dryingthat yields a powder of the active ingredient plus any additionaldesired ingredient front a previously sterile-filtered solution thereof.The proper fluidity of a solution can be maintained, for example, by theuse of a coating such as lecithin, by the maintenance of the requiredparticle size in the case of dispersion and by the use of surfactants.Prolonged absorption of injectable compositions can be brought about byincluding in the composition an agent that delays absorption, forexample, monostearate salts and gelatin.

Supplementary active compounds can also be incorporated into thecompositions. In certain embodiments, an antibody, or antigen-bindingportion thereof, for use in the methods of the invention is coformulatedwith and/or coadministered with one or more additional therapeuticagents. For example, an anti-hTNFα antibody or antibody portion of theinvention may be coformulated and/or coadministered with one or moreDMARD or one or more NSAID or one or more additional antibodies thatbind other targets (e.g., antibodies that bind other cytokines or thatbind cell surface molecules), one or more cytokines, soluble TNFαreceptor (see e.g., PCT Publication No. WO 94/06476) and/or one or morechemical agents that inhibit hTNFα production or activity (such ascyclohexane-ylidene derivatives as described in PCT Publication No. WO93/19751) or any combination thereof. Furthermore, one or moreantibodies of the invention may be used in combination with two or moreof the foregoing therapeutic agents. Such combination therapies mayadvantageously utilize lower dosages of the administered therapeuticagents, thus avoiding possible side effects, complications or low levelof response by the patient associated with the various monotherapies.

In one embodiment, the invention includes pharmaceutical compositionscomprising an effective amount of a TNFα antibody, or antigen-bindingportion thereof, and a pharmaceutically acceptable carrier, wherein theeffective amount of the TNFα antibody may be effective to treat aTNFα-related disorder, including, for example, Crohn's disease. In oneembodiment, the antibody or antibody portion is incorporated into apharmaceutical formulation as described in PCT/IB03/04502 and U.S.application Ser. No. 10/222,140, incorporated by reference herein. Thisformulation includes a concentration 50 mg/ml of the antibodyadalimumab, wherein one pre-filled syringe contains 40 mg of antibodyfor subcutaneous injection.

The antibodies, or antibody-portions, obtained using the methods of thepresent invention can be administered by a variety of methods known inthe art, although for many therapeutic applications, the preferredroute/mode of administration is subcutaneous injection. In anotherembodiment, administration is via intravenous injection or infusion. Aswill be appreciated by the skilled artisan, the mute and/or mode ofadministration will vary depending upon the desired results. In certainembodiments, the active compound may be prepared with a carrier thatwill protect the compound against rapid release, such as a controlledrelease formulation, including implants, transdermal patches, andmicroencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Manymethods for the preparation of such formulations are patented orgenerally known to those skilled in the art. See, e.g., Sustained andControlled Release Drug Delivery Systems, J. R. Robinson, ed., MarcelDekker, Inc., New York, 1978.

The antibodies, or antigen-binding portion thereof, obtained using themethods of the invention can also be administered in the form of proteincrystal formulations which include a combination of protein crystalsencapsulated within a polymeric carrier to form coated particles. Thecoated particles of the protein crystal formulation may have a sphericalmorphology and be microspheres of up to 500 micro meters in diameter orthey may have some other morphology and be microparticulates. Theenhanced concentration of protein crystals allows the antibody of theinvention to be delivered subcutaneously. In one embodiment, theantibodies of the invention are delivered via a protein delivery system,wherein one or more of a protein crystal formulation or composition, isadministered to a subject with a TNFα-related disorder. Compositions andmethods of preparing stabilized formulations of whole antibody crystalsor antibody fragment crystals are also described in WO 02/072636, whichis incorporated by reference herein. In one embodiment, a formulationcomprising the crystallized antibody fragments described inPCT/IB03/04502 and U.S. application Ser. No. 10/222,140, incorporated byreference herein, are used to treat a TNFα-related disorder using themultiple-variable dose methods of the invention.

In certain embodiments, an antibodies, or antigen-binding portionthereof, obtained using the methods of the invention may e orallyadministered, for example, with an inert diluent or an assimilableedible carrier. The compound (and other ingredients, if desired) mayalso be enclosed in a hard or soft shell gelatin capsule, compressedinto tablets, or incorporated directly into the subject's diet. For oraltherapeutic administration, the compounds may be incorporated withexcipients and used in the form of ingestible tablets, buccal tablets,troches, capsules, elixirs, suspensions, syrups, wafers, and the like.To administer a compound of the invention by other than parenteraladministration, it may be necessary to coat the compound with, orco-administer the compound with, a material to prevent its inactivation.

The pharmaceutical compositions of the invention may include a“therapeutically effective amount” or a “prophylactically effectiveamount” of an antibody or antigen-binding portion thereof of theinvention. A “therapeutically effective amount” refers to an amounteffective, at dosages and for periods of time necessary, to achieve thedesired therapeutic result. A therapeutically effective amount of theantibody, or antigen-binding portion thereof, may vary according tofactors such as the disease state, age, sex, and weight of theindividual, and the ability of the antibody, antibody portion, otherTNFα inhibitor to elicit a desired response in the individual. Atherapeutically effective amount is also one in which any toxic ordetrimental effects of the antibody, or antigen-binding portion thereof,are outweighed by the therapeutically beneficial effects. A“prophylactically effective amount” refers to an amount effective, atdosages and for periods of time necessary, to achieve the desiredprophylactic result. Typically, since a prophylactic dose is used insubjects prior to or at an earlier stage of disease, theprophylactically effective amount will be less than the therapeuticallyeffective amount.

Dosage regimens may be adjusted to provide the optimum desired response(e.g., a therapeutic or prophylactic response). For example, a singlebolus may be administered, several divided doses may be administeredover time or the dose may be proportionally reduced or increased asindicated by the exigencies of the therapeutic situation. It isespecially advantageous to formulate parenteral compositions in dosageunit form for ease of administration and uniformity of dosage. Dosageunit form as used herein refers to physically discrete units suited asunitary dosages for the mammalian subjects to be treated; each unitcomprising a predetermined quantity of active compound calculated toproduce the desired therapeutic effect in association with the requiredpharmaceutical carrier. The specification for the dosage unit forms ofthe invention are dictated by and directly dependent on (a) the uniquecharacteristics of the active compound and the particular therapeutic orprophylactic effect to be achieved, and (b) the limitations inherent inthe art of compounding such an active compound for the treatment ofsensitivity in individuals.

An exemplary, non-limiting range for a therapeutically orprophylactically effective amount of an antibody, or antigen-bindingportion thereof, is 10 to 200 mg, more preferably 20 to 160 mg, morepreferably 40 to 80 mg, and most preferably 80 mg. In one embodiment,the therapeutically effective amount of an antibody or, antigen-bindingportion thereof, is about 20 mg. In another embodiment, thetherapeutically effective amount of an antibody or portion thereof isabout 40 mg. In still another embodiment, the therapeutically effectiveamount of an antibody or, antigen-binding portion thereof, is about 80mg. In one embodiment, the therapeutically effective amount of anantibody or portion thereof for use in the methods of the invention isabout 120 mg. In yet another embodiment, the therapeutically effectiveamount of an antibody, or antigen-binding portion thereof, is about 160mg. Ranges intermediate to the above recited dosages. e.g. about 78.5 toabout 81.5; about 15 to about 25; about 30 to about 50; about 60 toabout 100; about 90 to about 150; about 120 to about 200, are alsointended to be part of this invention. For example, ranges of valuesusing a combination of any of the above recited values as upper and/orlower limits are intended to be included.

It is to be noted that dosage values may vary with the type and severityof the condition to be alleviated. It is to be further understood thatfor any particular subject, specific dosage regimens should be adjustedover time according to the individual need and the professional judgmentof the person administering or supervising the administration of thecompositions, and that dosage ranges set forth herein are exemplary onlyand are not intended to limit the scope or practice of the claimedcomposition.

Antibodies, or antibody-portions thereof, obtained using the methods ofthe invention may be administered on a biweekly dosing regimen asdescribed in WO 02/100330, a low dose regimen as described in WO04/037205, and a multiple variable dosing regimen as described in WO05/110452, each of which is incorporated by reference herein.

The invention also pertains to packaged pharmaceutical compositions,articles of manufacture, or kits comprising the antibody, orantigen-binding portion thereof, obtained using the process of theinvention. The article of manufacture may comprise an antibody, orantigen-binging portion thereof, obtained using the method of theinvention and packaging material. The article of manufacture may alsocomprise label or package insert indicating the formulation orcomposition comprising the antibody, or antigen-binding portion thereof,has a reduced level of HCP and/or procathepsin L. The article ofmanufacture may comprise a label or package insert contained within thepackaging material indicating that the adalimumab formulation comprisesno greater than about 70 ng/mg of HCP or a label or package insertcontained within the packaging material indicating that the adalimumabformulation comprises no greater than about 13 ng/mg. The article ofmanufacture may comprise a label or package insert contained within thepackaging material indicating that the adalimumab formulation comprisesno greater than about 5 ng HCP/mg adalimumab. The article of manufacturemay also comprise packaging material indicating that the adalimumabformulation comprises no greater a level of procathepsin L than thatindicated by a cathepsin L activity of about 3.0 RFU/s/mg adalimumab.

VII. Methods of Treatment

The invention a method of producing an HCP- or procathepsin L-reducedantibody preparation which can be used for inhibiting TNFα activity in asubject suffering from a disorder in which TNFα activity is detrimental.TNFα has been implicated in the pathophysiology of a wide variety ofdisorders (see e.g., Moeller, A., et al. (1991) Cytokine 2:162-169; U.S.Pat. No. 5,231,024 to Moeller et al.; European Patent Publication No.260 610 B1 by Moeller, A.). TNFα has been implicated in thepathophysiology of a wide variety of a TNFα-related disorders includingsepsis, infections, autoimmune diseases, transplant rejection andgraft-versus-host disease (see e.g., Moeller, A., et al. (1990) Cytokine2:162-169; U.S. Pat. No. 5,231,024 to Moeller et al.; European PatentPublication No. 260 610 B1 by Moeller, A., et al. Vasilli, P. (1992)Annu. Rev. Immunol. 10:411-452; Tracey, K. J. and Cerami, A. (1994)Annu. Rev. Med. 45:491-503). The invention a method of producing an HCP-or procathepsin L-reduced antibody preparation methods which arebeneficial for inhibiting TNFα activity in a subject suffering from aTNFα-related disorder, which method comprises administering to a subjectan initial induction dose and subsequently administering a treatmentdose of an antibody, or antigen-binding fragment thereof, such that TNFαactivity is inhibited. Preferably, the TNFα is human TNFα and thesubject is a human subject. In one embodiment, the TNFα inhibitor isadalimumab, also referred to as HUMIRA® (D2E7).

As used herein, the term “a disorder in which TNFα activity isdetrimental” is intended to include diseases and other disorders inwhich the presence of TNFα in a subject suffering from the disorder hasbeen shown to be or is suspected of being either responsible for thepathophysiology of the disorder or a factor that contributes to aworsening of the disorder. Accordingly, a disorder in which TNFαactivity is detrimental is a disorder in which inhibition of TNFαactivity is expected to alleviate the symptoms and/or progression of thedisorder. Such disorders may be evidenced, for example, by an increasein the concentration of TNFα in a biological fluid of a subjectsuffering from the disorder (e.g., an increase in the concentration ofTNFα in serum, plasma, synovial fluid, etc. of the subject), which canbe detected, for example, using an anti-TNFα antibody as describedabove. There are numerous examples of disorders in which TNFα activityis detrimental. The use of TNFα antibodies and antibody portionsobtained using methods of the invention for the treatment of specificdisorders is discussed further below:

A. Sepsis

Tumor necrosis factor has an established role in the pathophysiology ofsepsis, with biological effects that include hypotension, myocardialsuppression, vascular leakage syndrome, organ necrosis, stimulation ofthe release of toxic secondary mediators and activation of the clottingcascade (see e.g., Moeller, A., et al. (1990) Cytokine 2:162-169; U.S.Pat. No. 5,231,024 to Moeller et al.; European Patent Publication No.260 610 B1 by Moeller, A.; Tracey. K. J. and Cerami, A. (1994) Annu.Rev. Med. 45:491-503; Russell, D and Thompson. R. C. (1993) Curr. Opin.Biotech. 4:714-721). The multiple-variable dose methods of the inventioncan be used to treat sepsis in any of its clinical settings, includingseptic shock, endotoxic shock, gram negative sepsis and toxic shocksyndrome.

Furthermore, to treat sepsis, an anti-hTNFα antibody, or antibodyportion, obtained using the process of the invention can becoadministered with one or more additional therapeutic agents that mayfurther alleviate sepsis, such as an interleukin-inhibitor (such asthose described in PCT Publication Nos. WO 92/16221 and WO 92/17583),the cytokine interleukin-6 (see e.g., PCT Publication No. WO 93/11793)or an antagonist of platelet activating factor (see e.g., EuropeanPatent Application Publication No. EP 374 510). In a preferredembodiment, an anti-TNFα antibody or antibody portion is administered toa human subject within a subgroup of sepsis patients having a serum orplasma concentration of IL-6 above 500 pg/ml, and more preferably 1000pg/ml, at the time of treatment (see PCT Publication No. WO 95/20978 byDaum, L, et al.).

B. Autoimmune Diseases

Tumor necrosis factor has been implicated in playing a role in thepathophysiology of a variety of autoimmune diseases. For example, TNFαhas been implicated in activating tissue inflammation and causing jointdestruction in rheumatoid arthritis (see e.g., Moeller, A., et al.(1990) Cytokine 2:162-169; U.S. Pat. No. 5,231,024 to Moeller et al.;European Patent Publication No. 260 610 B1 by Moeller, A.; Tracey andCerami. supra; Arend, W. P. and Dayer, J-M. (1995) Arth. Rheum.38:151-160; Fava, R. A., et al. (1993) Clin. Exp. Immunol. 94:261-266).TNFα also has been implicated in promoting the death of islet cells andin mediating insulin resistance in diabetes (see e.g., Tracey andCerami, supra; PCT Publication No. WO 94/08609). TNFα also has beenimplicated in mediating cytotoxicity to oligodendrocytes and inductionof inflammatory plaques in multiple sclerosis (see e.g., Tracey andCerami, supra). TNFα also has been implicated in mediating cytotoxicityto oligodendrocytes and induction of inflammatory plaques in multiplesclerosis (see e.g., Tracey and Cerami, supra). Chimeric and humanizedmurine anti-hTNFα antibodies have undergone clinical testing fortreatment of rheumatoid arthritis (see e.g., Elliott. M. J., et al.(1994) Lancet 344:1125-1127; Elliot, M. J., et al. (1994) Lancet34:1105-1110; Rankin, E. C., et al. (1995) Br. J. Rheumatol.34:334-342).

TNFα antibodies, such as adalimumab, may be used to treat autoimmunediseases, in particular those associated with inflammation. Examples ofsuch autoimmune conditions include rheumatoid arthritis, rheumatoidspondylitis, osteoarthritis and gouty arthritis, allergy, multiplesclerosis, autoimmune diabetes, autoimmune uveitis and nephroticsyndrome. Other examples of autoimmune conditions include multisystemautoimmune diseases and autoimmune hearing loss. Typically, theantibody, or antibody portion, is administered systemically, althoughfor certain disorders, local administration of the antibody or antibodyportion at a site of inflammation may be beneficial (e.g., localadministration in the joints in rheumatoid arthritis or topicalapplication to diabetic ulcers, alone or in combination with acyclohexane-ylidene derivative as described in PCT Publication No. WO93/19751). TNFα inhibitors, including human antibodies, and antibodyportions such as D2E7, also can be administered with one or moreadditional therapeutic agents useful in the multiple-variable dosetreatment of autoimmune diseases, as discussed further below.

In one embodiment of the invention, a TNFα antibody obtained using themethods of the invention is used to treat autoimmune disorders such aslupus. Lupus is has been shown to be associated with TNF activity(Shvidel et al. (2002) Hematol J. 3:32; Studnicka-Benke et al. (1996) BrJ Rheumatol. 35:1067). The term “lupus” as used herein refers to achronic, inflammatory autoimmune disorder called lupus erythematosusthat may affect many organ systems including the skin, joints andinternal organs. Lupus is a general term which includes a number ofspecific types of lupus, including systemic lupus, lupus nephritis, andlupus cerebritis. In systemic lupus (SLE), the body's natural defensesare turned against the body and rogue immune cells attack the body'stissues. Antibodies may be produced that can react against the body'sblood cells, organs, and tissues. This reaction leads to immune cellsattacking the affected systems, producing a chronic disease. Lupusnephritis, also referred to as lupus glomerular disease, is kidneydisorder that is usually a complication of SLE, and is characterized bydamage to the glomerulus and progressive loss of kidney function. Lupuscerebritis refers to another complication of SLE, which is inflammationof the brain and/or central nervous system.

Another autoimmune disease which can be treated using a TNFα antibody isCrohn's disease, which is described in more detail below in theIntestinal Disorders Section.

C. Infectious Diseases

Tumor necrosis factor has been implicated in mediating biologicaleffects observed in a variety of infectious diseases. For example, TNFαhas been implicated in mediating brain inflammation and capillarythrombosis and infarction in malaria. TNFα also has been implicated inmediating brain inflammation, inducing breakdown of the blood-brainbarrier, triggering septic shock syndrome and activating venousinfarction in meningitis. TNFα also has been implicated in inducingcachexia, stimulating viral proliferation and mediating central nervoussystem injury in acquired immune deficiency syndrome (AIDS) Accordingly,antibodies, and antibody portions, directed against TNF, can be used fortreatment of infectious diseases, including bacterial meningitis (seee.g., European Patent Application Publication No. EP 585 705), cerebralmalaria, AIDS and AIDS-related complex (ARC) (see e.g., European PatentApplication Publication No. EP 230 574), as well as cytomegalovirusinfection secondary to transplantation (see e.g., Fietz et al. (1994)Transplantation 58:675). The antibodies, and antibody portions, of theinvention, also can be used to alleviate symptoms associated withinfectious diseases, including fever and myalgias due to infection (suchas influenza) and cachexia secondary to infection (e.g., secondary toAIDS or ARC).

D. Transplantation

Tumor necrosis factor has been implicated as a key mediator of allograftrejection and graft versus host disease (GVHD) and in mediating anadverse reaction that has been observed when the rat antibody OKT3,directed against the T cell receptor CD3 complex, is used to inhibitrejection of renal transplants (see e.g., Eason et al. (1995)Transplantation 59:300; Suthanthiran and Strom (1994) New Engl. J. Med.331:365). Accordingly, the antibodies, and antibody portions, of theinvention, can be used to inhibit transplant rejection usingmultiple-variable dose treatment, including rejections of allografts andxenografts and to inhibit GVHD. Although the antibody or antibodyportion may be used alone, more preferably it is used in combinationwith one or more other agents that inhibit the immune response againstthe allograft or inhibit GVHD. For example, in one embodiment, anantibody or antibody portion of the invention is used in combinationwith OKT3 to inhibit OKT3-induced reactions. In another embodiment, anantibody or antibody portion of the invention is used in combinationwith one or more antibodies directed at other targets involved inregulating immune responses, such as the cell surface molecules CD25(interleukin-2 receptor-α), CD11a (LFA-1), CD54 (ICAM-1), CD4, CD45,CD28/CTLA4, CD80 (B7-1) and/or CD86 (B7-2). In yet another embodiment,an antibody or antibody portion of the invention is used in combinationwith one or more general immunosuppressive agents, such as cyclosporin Aor FK506.

E. Malignancy

Tumor necrosis factor has been implicated in inducing cachexia,stimulating tumor growth, enhancing metastatic potential and mediatingcytotoxicity in malignancies. Accordingly, antibodies, and antibodyportions, which directed against TNF, can be used in the treatment ofmalignancies, wherein treatment inhibits tumor growth or metastasisand/or alleviates cachexia secondary to malignancy. The antibody, orantibody portion, may be administered systemically or locally to thetumor site.

F. Pulmonary Disorders

Tumor necrosis factor has been implicated in the pathophysiology ofadult respiratory distress syndrome (ARDS), including stimulatingleukocyte-endothelial activation, directing cytotoxicity to pneumocytesand inducing vascular leakage syndrome. The antibody obtained using themethods of the invention may be used to treat various pulmonarydisorders, including adult respiratory distress syndrome (see e.g., PCTPublication No. WO 91/04054), shock long, chronic pulmonary inflammatorydisease, pulmonary sarcoidosis, pulmonary fibrosis and silicosis. Theantibody, or antibody portion, may be administered systemically orlocally to the lung surface, for example as an aerosol. An antibody, orantibody portion, also can be administered with one or more additionaltherapeutic agents useful in the treatment of pulmonary disorders, asdiscussed further below.

Other examples of pulmonary disorders in which TNFα has been implicatedin the pathophysiology include idiopathic interstitial lung disease andchronic obstructive airway disorders (see e.g., Piquet et al. (1989) JExp Med. 170:655; Whyte et al. (2000) Am J Respir Crit Care Med.162:755; Anticevich et al. (1995) Eur J Pharmacol. 284:221). Theinvention further provides methods for treating TNFα activity in asubject suffering from such a pulmonary disorder, which method comprisesadministering to the subject an antibody, or antibody portion, such thatTNFα activity in the subject suffering from idiopathic interstitial lungdisease or a chronic obstructive airway disorder is inhibited. Examplesof idiopathic interstitial lung diseases and chronic obstructive airwaydisorders in which TNFα activity is detrimental are discussed furtherbelow.

1. Idiopathic Interstitial Lung Disease

In one embodiment, the TNFα antibody obtained using the method of theinvention is used to treat subjects who have an idiopathic interstitiallung disease. The term “idiopathic pulmonary fibrosis” or “IPF” refersto a group of disorders characterized by inflammation and eventuallyscarring of the deep lung tissues, leading to shortness of breath. Thescarring of the alveoli (air sacs) and their supporting structures (theinterstitium) in IPF eventually leads to a loss of the functionalalveolar units and a reduction of the transfer of oxygen from air toblood. IPF is also referred to as diffuse parenchymal lung disease;alveolitis; cryptogenic fibrosing alveolitis (CFA); idiopathic pulmonarypneumonitis (IPP); and usual interstitial pneumonitis (IP). IPF is oftenused synonymously with UIP (“IPF/UIP”) because UIP is the most commoncellular pattern seen in the pathologic diagnosis of IPF.

Idiopathic interstitial lung diseases affect the lungs in three ways;first, the lung tissue is damaged in some known or unknown way; second,the walls of the air sacs in the lung become inflamed; and finally,scarring (or fibrosis) begins in the interstitium (or tissue between theair sacs), and the lung becomes stiff. Examples of idiopathicinterstitial lung diseases include idiopathic pulmonary fibrosis (IPF).Tumor necrosis factor has been implicated in the pathophysiology ofidiopathic pulmonary fibrosis (IPF) (see Piquet et al. (1989) J Exp Med.170:655; Whyte et al. (2,0)) Am J Respir Crit Care Med 162:755 Corbettet al. (2002) Am J Respir Crit Care Med. 165:690). For example, it hasbeen found that IPF patients have increased levels of TNF expression inmacrophages and in type II epithelial cells (Piquet et al. (1993) Am JPathol 143:651; Nash et al. (1993) Histopathology 22:343; Zhang et al.(1993) J Immunol 150:4188). Certain genetic polymorphisms are alsoassociated with increased TNF expression, and are implicated in playinga role in IPF and silicosis (Whyte et al., supra; Corbett et al.,supra).

Patients with IPF often exhibit certain symptoms, including a dry cough,chest pain, and/or shortness of breath. Commonly used drugs for thetreatment of IPF are prednisone and cytoxan, although only a fraction ofpatients improve with continued use of these drugs (American ThoracicSociety (2000) Am J. Respir. Crit. Care Med. 161:646). Oxygenadministration and transplantation of the lung are other choices fortreatment. In one embodiment, antibodies obtained through the methods ofthe invention may be used in combination with another therapeutic agent,for example oxygen, for the treatment of idiopathic pulmonary fibrosis.

Examples of animal models used to study idiopathic interstitial lungdisease and chronic obstructive airway disorders include ovalbumin (OVA)induced allergic asthma mice and cigarette smoke induced chronicobstructive pulmonary disease mice (see Hessel et al. (1995) Eur JPharmacol. 293:401; Keast et al. (1981) J. Pathol. 135:249).

2. Chronic Obstructive Airway Disorder

In one embodiment, a TNFα antibody is used to treat a subject who has achronic obstructive airflow disorder. In these diseases, airflowobstruction may be chronic and persistent or episodic and recurrent.Airflow obstruction is usually determined by forced expiratoryspirometry, which is the recording of exhaled volume against time duringa maximal expiration. In a subject who does not have an obstructedairflow, a full forced expiration usually takes between 3 and 4 seconds.In a patient with chronic obstructive airflow disorder, wherein airflowis obstructed, it usually takes up to 15 to 20 seconds and may belimited by breath-holding time. The normal forced expiratory volume inthe first second of expiration (FEV₁) is easily measured and accuratelypredicted on the basis of age, sex, and height. The ratio of FEV₁ toforced vital capacity (FEV₁/FVC) normally exceeds 0.75. Recordingairflow against volume during forced expiration and a subsequent forcedinspiration—the flow-volume loop—is also useful, mainly fordistinguishing upper from lower airway narrowing. Examples of chronicobstructive airway disorders are described below.

a. Asthma

Tumor necrosis factor has been implicated in the pathophysiology ofasthma, (Anticevich et al. (1995) Eur J Pharmacol. 284:221; Thomas etal. 1995. Am J Respir Crit Care Med. 152:76; Thomas and Heywood (2002)Thorax. 57:774). For example, acute asthma attacks have been found to beassociated with pulmonary neutrophilia and elevated BAL TNF levels(Ordonez et al. (2000) Am J Respir Crit Care Med 161:1185). It has beenfound that the severity of asthma symptoms correlates with endotoxinlevels in house dust. In rats, anti-TNF antibodies reducedendotoxin-induced airway changes (Kips et al. (1992) Am Rev Respir Dis145:332).

The term “asthma” as used herein, refers to a disorder in whichinflammation of the airways causes airflow into and out of the lungs tobe restricted. Asthma is also referred to as bronchial asthma, exerciseinduced asthma—bronchial, and reactive airways disease (RAD). In someinstances, asthma is associated with allergies and/or is familial.Asthma includes a condition which is characterized by widespreadfluctuations in the diameter or caliber of bronchial airways over shortperiods of time, resulting in changes in lung function. The resultingincreased resistance to air flow produces symptoms in the affectedsubject, including breathlessness (dyspnea), chest constriction or“tightness,” and wheezing.

Patients with asthma are characterized according to NIH guidelines, aredescribed as mild intermittent, mild persistent, moderate persistent,and severe persistent (see NAEPP Expert Panel Report Guidelines for theDiagnosis and Management of Asthma-Update on Selected Topics 2002. JACI2002; 110: S141-S209; (Guidelines for the Diagnosis and Management ofAsthma. NIH Publication 97-4051, July 1997). Patients diagnosed withmoderate persistent asthma are often treated with inhaledcorticosteroids. Patients diagnosed with severe persistent asthma areoften treated with high dose inhaled corticosteroids and p.o.corticosteroids.

b. Chronic obstructive pulmonary disease. (COPD)

Tumor necrosis factor has been implicated in the pathophysiology ofchronic obstructive pulmonary disease, (Keatings (2000) Chest. 118:971;Sakao et al. (2001) Am J Respir Crit Care Med. 163:420; Sakao et al.(2002) Chest. 122:416). The term “chronic obstructive pulmonary disease”or “COPD” as used interchangeably herein, refers to a group of lungdiseases characterized by limited airflow with variable degrees of airsack enlargement and lung tissue destruction. The term COPD includeschronic bronchitis (mucous hypersecretion with goblet cell submucosalgland hyperplasia), chronic obstructive bronchitis, or emphysema(destruction of airway parenchyma), or combinations of these conditions.Emphysema and chronic bronchitis are the most common forms of chronicobstructive pulmonary disease. COPD is defined by irreversible airflowobstruction.

In COPD, chronic inflammation leads to fixed narrowing of small airwaysand lung parenchyma and alveolar wall destruction (emphysema). This ischaracterized by increased numbers of alveolar macrophages, neutrophils,and cytotoxic T lymphocytes, and the release of multiple inflammatorymediators (lipids, chemokines, cytokines, growth factors). Thisinflammation leads to fibrosis with a narrowing of the small airways andlung parenchymal destruction. There is also a high level of oxidativestress, which may amplify this inflammation.

G. Intestinal Disorders

Tumor necrosis factor has been implicated in the pathophysiology ofinflammatory bowel disorders including Crohn's disease (see e.g., Tracyet al. (1986) Science 234:470; Sun et al. (1988) J. Clin. Invest.81:1328; MacDonald et al. (1990) Clin. Exp. Immunol. 81:301). Chimericmurine anti-hTNFα antibodies have undergone clinical testing fortreatment of Crohn's disease (van Dullemen et al. (1995)Gastroenterology 109:129). The invention includes treatment comprisingadministering a TNFα antibody obtained using the method of the inventionto treat intestinal disorders, such as idiopathic inflammatory boweldisease, using human antibodies, or antigen-binding fragments thereof.Idiopathic inflammatory bowel disease includes two syndromes, Crohn'sdisease and ulcerative colitis. In one embodiment, an antibody obtainedusing the method of the invention is also used to treat disorders oftenassociated with IBD and Crohn's disease. The term “inflammatory boweldisorder (IBD)-related disorder” or “Crohn's disease-related disorder,”as used interchangeably herein, is used to describe conditions andcomplications commonly associated with IBD) and Crohn's disease.

The invention includes a multiple-variable dose regimen comprisingadministering a TNFα antobody to treat Crohn's disease. The treatment ofCrohn's disease is based on location, extent, and seventy of disease.Pharmacologic interventions include anti-inflammatory agents(aminosalicylates and corticosteroids) and immunomodulatory agents(azathioprine and 6-mercaptopurine [6-MP], cyclosporine, methotrexate[MTX], antibiotic agents, and biologic agents) C-reactive protein (CRP)and erythrocyte sedimentation rate (ESR) levels reflect non-specificacute phase reactions. Endoscopy is a primary means of diagnosingCrohn's disease. Radiologic features of Crohn's disease are shown bybarium examination includes mucosal edema, aphthous and linearulcerations, asymmetrical narrowing and strictures, and separation ofadjacent loops of bowel caused by mesenteric thickening. Abnormalitiesare focal and asymmetric. The primary histologic lesion is an aphthousulcer. Subjects with Crohn's disease can be evaluated using the Crohn'sDisease Activity Index (CDAI), which is a standard measure of theseverity of the disease with higher scores indicating more severedisease activity.

Examples of Crohn's disease-related disorders which can be treated usingthe methods of the invention include fistulas in the bladder, vagina,and skin; bowel obstructions; abscesses; nutritional deficiencies;complications from corticosteroid use; inflammation of the joints;erythem nodosum; pyoderma gangrenosum; and lesions of the eye. Otherdisorders commonly associated with Crohn's disease includeCrohn's-related arthralgias, fistulizing Crohn's, indeterminant colitis,and pouchitis.

H. Cardiac Disorders

An antibody, or antigen-binding fragment thereof, obtained using themethod of the invention also can be used to treat in of various cardiacor coronary disorders, including ischemia of the heart. (see e.g.,European Patent. Application Publication No. EP 453 898) and heartinsufficiency (weakness of the heart muscle) (see e.g., PCT PublicationNo. WO 94/20139). TNFα has also been implicated in the pathophysiologyof restenosis (see e.g., Clausell et al. (1994), supra; Medall et al.(1997) Heart 78:273).

As used herein, the term “a cardiac disorder in which TNFα activity isdetrimental” is intended to include coronary and cardiovascular diseasesin which the presence of TNFα in a subject suffering from the disorderhas been shown to be or is suspected of being either responsible for thepathophysiology of the disorder or a factor that contributes to aworsening of the disorder, including cardiovascular disorders, e.g.,restenosis. The term “cardiovascular disorder” or “coronary disorder” asused interchangeably herein, refers to any disease, disorder, or stateinvolving the cardiovascular system, e.g., the heart, the blood vessels,and/or the blood. A coronary disorder is generally characterized by anarrowing of the blood vessels that supply blood and oxygen to the heart(coronary arteries). Coronary disease may result from the build up offatty material and plaque. As the coronary arteries narrow, the flow ofblood to the heart can slow or stop. Coronary disorders of the inventioncan apply to any abnormality of an artery, whether structural,histological, biochemical or any other abnormality. An example ofcoronary heart disease is restenosis. In one embodiment, a coronarydisorder refers to any disease, disorder, or state involving thecardiovascular system excluding ischemia of the heart and heartinsufficiency.

Coronary disorders in which TNFα activity is detrimental often resultfrom a blockage in an artery. Such a blockage can be caused by a clot,which usually forms in a coronary artery that has been previouslynarrowed from changes usually related to atherosclerosis. For example,if the atherosclerotic plaque inside the arterial wall cracks, it cantrigger the formation of a thrombus, or clot. Such disorders may beevidenced, for example, by an increase in the concentration of TNFα in abiological fluid of a subject suffering from the disorder (e.g., anincrease in the concentration of TNFα in serum, plasma, synovial fluid,etc. of the subject), which can be detected, for example, using ananti-TNFα antibody as described above. A coronary disorder can be alsocaused by an imbalance in arterial pressure, a malfunction of the heart,or an occlusion of a blood vessel, e.g., by a thrombus. Coronarydisorders includes both coronary artery disease and peripheral vasculardisease.

There are numerous examples of cardiac disorders in which TNFα activityis detrimental, including restenosis. The use of the antibodies,antibody portions, for treatment of specific coronary disorders isdiscussed further below. In certain embodiments, an antibody, antibodyportion, is administered to the subject in combination with anothertherapeutic agent, as described below.

Antibodies obtained using methods of the invention may also be used forinhibiting TNFα activity in a subject with a cardiac disorder. Theinvention provides methods for inhibiting or decreasing TNFα activity ina subject with a coronary disorder, comprising administering to thesubject an antibody, or antibody portion, or other TNFα inhibitor of theinvention such that TNFα activity in the subject is inhibited ordecreased. Preferably, the TNFα is human TNFα and the subject is a humansubject. Alternatively, the subject can be a mammal expressing a TNFαwith which an antibody of the invention cross-reacts. Still further thesubject can be a mammal into which has been introduced hTNFα (e.g., byadministration of hTNFα or by expression of an hTNFα transgene). Anantibody of the invention can be administered to a human subject fortherapeutic purposes.

Moreover, an anti body of the invention can be administered to anon-human mammal expressing a TNFα with which the antibody cross-reacts(e.g., a primate, pig or mouse) for veterinary purposes or as an animalmodel of human disease. Regarding the latter, such animal models may beuseful for evaluating the multiple-variable dose therapeutic efficacy(e.g., testing of dosages and time courses of administration). Commonlyused animal models for studying coronary disorders, includingrestenosis, include the rat or mouse carotid artery ligation model andthe carotid artery injury model (Ferns et al. (1991) Science 253:1129;Clowes et al. (1983) Lab. Invest 49:208; Lindner et al. (1993) Circ Res.73:792). In the carotid artery ligation model, arterial blood flow isdisrupted by ligation of the vessel near the distal bifurnation. Asdescribed in Clowes et al., the carotid artery injury model is performedsuch that the common carotid artery is denuded of endothelium by theintraluminal passage of a balloon catheter introduced through theexternal carotid artery. At 2 weeks, the carotid artery is markedlynarrowed due to smooth muscle cell constriction, but between 2 and 12weeks the intimal doubles in thickness leading to a decrease in luminalsize. Any of these models can be used to determine the potentialtherapeutic action of the TNFα antibodies of the invention in theprevention and treatment of restenosis in humans.

The invention includes treatment of cardiovascular disorders in whichTNFα activity is detrimental, wherein inhibition of TNFα activity isexpected to alleviate the symptoms and/or progression of the coronarydisease or to prevent the coronary disease. Subjects suffering from orat risk of developing coronary disorders can be identified throughclinical symptoms. Clinical symptoms in coronary disease often includechest pain, shortness of breath, weakness, fainting spells, alterationsin consciousness, extremity pain, paroxysmal nocturnal dyspnea,transient ischemic attacks and other such phenomena experienced by thepatient. Clinical signs of coronary disease can also include EKGabnormalities, altered peripheral pulses, arterial bruits, abnormalheart sounds, rates and wheezes, jugular venous distention, neurologicalalterations and other such findings discerned by the clinician. Coronarydisorders may also be evidenced, for example, by an increase in theconcentration of TNFα in a biological fluid of a subject suffering fromthe disorder (e.g., an increase in the concentration of TNFα in serum,plasma, synovial fluid, etc. of the subject).

Examples of a cardiovascular disorder include, but are not limited to,coronary artery disease, angina pectoris, myocardial infarction,cardiovascular tissue damage caused by cardiac arrest, cardiovasculartissue damage caused by cardiac bypass, cardiogenic shock, andhypertension, atherosclerosis, coronary artery spasm, coronary arterydisease, valvular disease, arrhythmias, and cardiomyopathies. The use ofthe antibodies, antibody portions, for treatment of specificcardiovascular diseases are discussed further below. In certainembodiments, the antibody, antibody portion, is administered to thesubject in combination with another therapeutic agent, as describedbelow.

1. Restenosis

The term “restenosis” as used herein refers to the recurrence ofstenosis, which is the narrowing or constriction of an artery.Restenosis often occurs as a preocclusive lesion that develops followinga reconstructive procedure in a diseased blood vessel. The term is notonly applied to the recurrence of a pre-existing stenosis, but also topreviously normal vessels that become partially occluded followingvascular bypass. In another embodiment, the invention provides a methodof treating restenosis comprising administering the antibody, or antigenbinding portion thereof, obtained using the invention to a subject whohas or is at risk of developing restenosis.

TNFα has been implicated in the pathophysiology of restenosis (see Zhouet al. (2002) Atherosclerosis. 161:153; Javed et al. (2002) Exp and MolPathol 73:104). For example, in the murine wire carotid model. TNF−/−mice demonstrated a seven-fold reduction in initial hyperplasia comparedto wild type mice (Zimmerman et al. (2002) Am J Phsiol Regul Integr CompPhysiol 283:R505). Restenosis can occur as the result of any type ofvascular reconstruction, whether in the coronary vasculature or in theperiphery (Colburn and Moore (1998) Myointimal Hyperplasia pp. 690-709in Vascular Surgery: A Comprehensive Review Philadelphia; Saunders). Forexample, studies have reported symptomatic restenosis rates of 30-50%following coronary angioplasties (see Berk and Harris (1995) Adv.Intern. Med. 40:455). After carotid endarterectomies, as a furtherexample, 20% of patients studied had a luminal narrowing greater than50% (Clagett et al. (1986) J. Vasc. Surg. 3:10). Restenosis is evidencedin different degrees of symptomatology which accompany preocclusivelesions in different anatomical locations, due to a combination offactors including the nature of the vessels involved, the extent ofresidual disease, and local hemodynamics.

“Stenosis,” as used herein refers to a narrowing of an artery as seen inocclusive disorder or in restenosis. Stenosis can be accompanied bythose symptoms reflecting a decrease in blood flow past the narrowedarterial segment, in which case the disorder giving rise to the stenosisis termed a disease (i.e., occlusive disease or restenosis disease).Stenosis can exist asymptotically in a vessel, to be detected only by adiagnostic intervention such as an angiography or a vascular lab study.

The antibodies obtained using the method of the invention may be used totreat a subject suffering from or at risk of developing restenosis. Asubject at risk of developing restenosis includes a subject who hasundergone PTCA. The subject may have also had a stent inserted toprevent restenosis. The TNFα antibody can be used alone or incombination with a stent to prevent the re-occurrence of stenosis in asubject suffering from cardiovascular disease.

2. Congestive Heart Failure

TNFα has been implicated in the pathophysiology of congestive heartfailure (see Zhou et al. (2002) Atherosclerosis 161:153). Serum levelsof TNFα are elevated in patients with congestive heart failure in amanner which is directly proportional to the severity of the disease(Levine et al. (1990) N Engl J Med 323:236; Torre-Amione et al. (1996) JAm Coll Cardiol 27:1201) In addition, inhibitors of TNFα have also beenshown to improve congestive heart failure symptoms (Chung et al. (2003)Circulation 107:3133).

As used herein, the term “congestive heart failure” includes a conditioncharacterized by a diminished capacity of the heart to supply the oxygendemands of the body. Symptoms and signs of congestive heart failureinclude diminished blood flow to the various tissues of the body,accumulation of excess blood in the various organs, e.g., when the heartis unable to pump out the blood returned to it by the great veins,exertional dyspnea, fatigue, and/or peripheral edema, e.g., peripheraledema resulting from left ventricular dysfunction. Congestive heartfailure may be acute or chronic. The manifestation of congestive heartfailure usually occurs secondary to a variety of cardiac or systemicdisorders that share a temporal or permanent loss of cardiac function.Examples of such disorders include hypertension, coronary arterydisease, valvular disease, and cardiomyopathies, e.g., hypertrophic,dilative, or restrictive cardiomyopathies.

A “subject who has or is suffering from congestive heart failure” is asubject who has a disorder involving a clinical syndrome of diverseetiologies linked by the common denominator of impaired heart pumping inwhich the heart cannot pump blood commensurate with the requirements ofthe metabolizing tissues, or can do so only from an elevated fillingpressure. A “subject at risk of developing congestive heart failure” isa subject who has a propensity of developing congestive heart failurebecause of certain factors affecting the cardiovascular system of thesubject. It is desirable to reduce the risk of or prevent thedevelopment of congestive heart failure in these subjects. The phrase“with congestive heart failure” includes patients who are at risk ofsuffering from this condition relative to the general population, eventhough they may not have suffered from it yet, by virtue of exhibitingrisk factors. For example, a patient with untreated hypertension may nothave suffered from congestive heart failure, but is at risk because ofhis or her hypertensive condition. In one embodiment of the invention,the antibody adalimumab is used to treat a subject at risk of developingcongestive heart failure.

3. Acute Coronary Syndromes

TNFα L has been implicated in the pathophysiology of acute coronarysyndromes (see Libby (1995) Circulation 91:2844). Acute coronarysyndromes include those disorders wherein the subject experiences paindue to a blood flow restriction resulting in not enough oxygen reachingthe heart. Studies have found that TNFα plays a role in acute coronarysyndromes. For example, in a novel rat heterotopic cardiactransplantation-coronary ligation model capable of inducing myocardialinfarction in the absence of downstream hemodynamic effects,administration of chimeric soluble TNFα receptor (sTNFR) abolishedtransient LV remodeling and dysfunction (Nakamura, et al. (2003) J.Cardiol. 41:41). It was also found that direct injection of an sTNFRexpression plasmid to the myocardium, resulted in a reduction in theinfarction size in acute myocardial infarction (AMI) experimental rats(Sugano et al. (2002) FASEB J 16:1421).

In one embodiment, a TNFα antibody is used for the treatment orprevention of an acute coronary syndrome in a subject, wherein the acutecoronary syndrome is a myocardial infarction or angina.

As used herein, the term “myocardial infarction” or “MI” refers to aheart attack. A myocardial infarction involves the necorsis or permanentdamage of a region of the heart due to an inadequate supply of oxygen tothat area. This necrosis is typically caused by an obstruction in acoronary artery from either atherosclerosis or an embolis. MIs which aretreated by the TNFα antibody obtained using the methods of the inventioninclude both Q-wave and non-Q-wave myocardial infarction. Most heartattacks are caused by a clot that blocks one of the coronary arteries(the blood vessels that bring blood and oxygen to the heart muscle). Forexample, a clot in the coronary artery interrupts the flow of blood andoxygen to the heart muscle, leading to the death of heart cells in thatarea. The damaged heart muscle permanently loses its ability tocontract, and the remaining heart muscle needs to compensate for it. AnMI can also be caused by overwhelming stress in the individual.

The term “angina” refers to spasmodic, choking, or suffocative pain, andespecially as denoting angina pectoris which is a paroxysmal thoracicpain due, most often, to anoxia of the myocardium. Angina includes bothvariant angina and exertional angina. A subject having angina hasischemic heart disease which is manifested by sudden, severe, pressingsubstemal pain that often radiates to the left shoulder and along theleft arm. TNFα has been implicated in angina, as TNFα levels areupregulated in patients with both MI and stable angina (Balbay et al.(2001) Angiology 52109).

4. Artherosclerosis

“Atherosclerosis” as used herein refers to a condition in which fattymaterial is deposited along the walls of arteries. This fatty materialthickens, hardens, and may eventually block the arteries.Atherosclerosis is also referred to arteriosclerosis, hardening of thearteries, and arterial plaque buildup. Polyclonal antibodies directedagainst TNFα have been shown to be effective at neutralizing TNFαactivity resulting in inflammation and restenosis in the rabbitatherosclerotic model (Zhou et al., supra). Accordingly, a TNFα □antibody may be used to treat or prevent subjects afflicted with or atrisk of having atherosclerosis.

5. Cardiomyopathy

The term “cardiomyopathy” as used herein is used to define diseases ofthe myocardium wherein the heart muscle or myocardium is weakened,usually resulting in inadequate heart pumping. Cardiomyopathy can becaused by viral infections, heart attacks, alcoholism, long-term, severehypertension (high blood pressure), or by autoimmune causes.

In approximately 75-80% of heart failure patients coronary arterydisease is the underlying cause of the cardiomyopathy and is designated“ischemic cardiomyopathy.” Ischemic cardiomyopathy is caused by heartattacks, which leave scars in the heart muscle or myocardium. Theaffected myocardium is then unable to contribute to the heart pumpingfunction. The larger the scars or the more numerous the heart attacks,the higher the chance there is of developing ischemic cardiomyopathy.

Cardiomyopathies that are not attributed to underlying coronary arterydisease, and are designated “non-ischemic cardiomyopathies.”Non-ischemic cardiomyopathies include, but are not limited to idiopathiccardiomyopathy, hypertrophic cardiomyopathy, alcoholic cardiomyopathy,dilated cardiomyopathy, peripartum cardiomyopathy, and restrictivecardiomyopathy.

I. Spondyloarthropathies

TNFα has been implicated in the pathophysiology of a wide variety ofdisorders, including inflammatory diseases such as spondyloarthopathies(see e.g., Moeller et al. (1990) Cytokine 2:1.62; U.S. Pat. No.5,231,024; European Patent Publication No. 260 610). The inventionprovides multiple-variable dose methods for inhibiting TNFα activity ina subject suffering from a spondyloarthropathy, which method comprisesadministering to the subject an antibody, antibody portion, such thatTNFα activity in the subject suffering from a spondyloarthropathy isinhibited.

As used herein, the term “spondyloarthropathy” or“spondyloarthropathies” is used to refer to any one of several diseasesaffecting the joints of the spine, wherein such diseases share commonclinical, radiological, and histological features. A number ofspondyloarthropathies share genetic characteristics, i.e. they areassociated with the HLA-B27 allele. In one embodiment, the termspondyloarthropathy is used to refer to any one of several diseasesaffecting the joints of the spine, excluding ankylosing spondylitis,wherein such diseases share common clinical, radiological, andhistological features. Examples of spondyloarthropathies includeankylosing spondylitis, psoriatic arthritis/spondylitis, enteropathicarthritis, reactive arthritis or Reiter's syndrome, and undifferentiatedspondyloarthropathies. Examples of animal models used to studyspondyloarthropathies include ank/ank transgenic mice. HLA-B27transgenic rats (see Taurog et al. (1998) The Spondylarthritides.Oxford, Oxford University Press).

The multiple-variable dose methods of the invention can also be used totreat subjects who are at risk of developing a spondyloarthropathy usingmultiple-variable dose methods. Examples of subjects who are at risk ofhaving spondyloarthropathies include humans suffering from arthritis.Spondyloarthropathies can be associated with other forms of arthritis,including rheumatoid arthritis. In one embodiment of the invention,antibodies are used in muitiple-variable dose methods to treat a subjectwho suffers from a spondyloarthropathy associated with rheumatoidarthritis. Examples of spondyloarthropathies which can be treated with aTNFα antibody are described below:

1. Ankylosing Spondylitis (AS)

Tumor necrosis factor has been implicated in the pathophysiology ofankylosing spondylitis (see Verjans et al. (1991) Arthritis Rheum.34:486; Verjans et al. (1994) Clin Exp Immunol. 97:45; Kaijtzel et al.(1999) Hum Immunol. 60:140). Ankylosing spondylitis (AS) is aninflammatory disorder involving inflammation of one or more vertebrae.AS is a chronic inflammatory disease that affects the axial skeletonand/or peripheral joints, including joints between the vertebrae of thespine and sacroiliac joints and the joints between the spine and thepelvis. AS can eventually cause the affected vertebrae to fuse or growtogether. Spondyarthropathies, including AS, can be associated withpsoriatic arthritis (PsA) and/or inflammatory bowel disease (IBD),including ulcerative colitis and Crohn's disease.

Early manifestations of AS can be determined by radiographic tests,including CT scans and MRI scans. Early manifestations of AS ofteninclude scroillitis and changes in the sacroliae joints as evidenced bythe blurring of the cortical margins of the subchrondral bone, followedby erosions and sclerosis. Fatigue has also been noted as a commonsymptom of AS (Duffy et al. (2002) ACR 66th Annual Scientific MeetingAbstract). Accordingly, multiple-variable dose methods comprisingadministering an antibody, or antigen-binding fragment thereof, of theinvention can be used to treat AS.

In one embodiment, the multiple-variable dose method of the invention isused to treat a spondyloarthropathy associated with IBD, including AS.AS is often treated with nonsteroidal anti-inflammatory medications(NSAIDs), such as aspirin or indomethacin. Accordingly, a TNFα antibodyused in the multiple-variable dose method of the invention may also beadministered in combination with agents commonly used to reduceinflammation and pain commonly associated with ankylosing spondylitis.

2. Psoriatic Arthritis

Tumor necrosis factor has been implicated in the pathophysiology ofpsoriatic arthritis (PsA) (Partsch et al. (1998) Ann Rheum Dis. 57:691;Ritchlin et al. (1998) J Rheumatol. 25:1544). As referred to herein,psoriatic arthritis or psoriasis associated with the skin, refers tochronic inflammatory arthritis which is associated with psoriasis, whichis a common chronic skin condition that causes red patches on the body.About 1 in 20 individuals with psoriasis will develop arthritis alongwith the skin condition, and in about 75% of cases, psoriasis precedesthe arthritis. PsA exhibits itself in a variety of ways, ranging frommild to severe arthritis, wherein the arthritis usually affects thefingers and the spine. When the spine is affected, the symptoms aresimilar to those of ankylosing spondylitis, as described above. The TNFαantibody, or antigen-binding fragment thereof, obtained using theinvention can be used for treatment of PsA.

PsA is sometimes associated with arthritis mutilans. Arthritis mutilansrefers to a disorder which is characterized by excessive bone erosionresulting in a gross, erosive deformity which mutilates the joint. Inone embodiment, antibodies obtained using the method of the inventionare used to treat arthritis mutilans.

3. Reactive Arthritis/Reiter's Syndrome

Tumor necrosis factor has been implicated in the pathophysiology ofreactive arthritis, which is also referred to as Reiter's syndrome(Braun et al. (1999) Arthritis Rheum. 42(10):2039). Reactive arthritis(ReA) refers to arthritis which complicates an infection elsewhere inthe body, often following enteric or urogenital infections. ReA is oftencharacterized by certain clinical symptoms, including inflammation ofthe joints (arthritis), urethritis, conjunctivitis, and lesions of theskin and mucous membranes. In addition, ReA can occurs followinginfection with a sexually transmitted disease or dysenteric infection,including chlamydia, campylobacter, salmonella, or yersinia.Accordingly, antibodies obtained using the method of the invention maybe used to treat ReA.

4. Undifferentiated Spondyloarthropathies

In one embodiment, antibodies obtained using methods of the inventionare used to treat subjects suffering from undifferentiatedspondyloarthropathies (see Zeidler et al. (1992) Rheum Dis Clin NorthAm. 18:187). Other terms used to describe undifferentiatedspondyloarthropathies include seronegative oligoarthritis andundifferentiated oligoarthritis. Undifferentiated spondyloarthropathies,as used herein, refers to a disorder wherein the subject demonstratesonly some of the symptoms associated with a spondyloarthropathy. Thiscondition is usually observed in young adults who do not have IBD,psoriasis, or the classic symptoms of AS or Reiter's syndrome. In someinstances, undifferentiated spondyloarthropathies may be an earlyindication of AS. In one embodiment, the invention comprisesadministering a TNFα antibody, or antigen-binding fragment thereof,obtained using the claimed process to treat undifferentiatedspondyloarthropathies.

J. Metabolic Disorders

TNFα has been implicated in the pathophysiology of a wide variety ofdisorders, including metabolic disorders, such as diabetes and obesity(Spiegelman and Hotamisligil (1993) Cell 73:625; Chu et al. (200) Int JObes Relat Metab Disord. 24:1085; Ishii et al. (2000) Metabolism.49:1616). The term “metabolic disorder,” as used herein, refers todiseases or disorders which affect how the body processes substancesneeded to carry out physiological functions. Examples of metabolicdisorders include, but are not limited to, diabetes and obesity. In oneembodiment of the invention, the term “metabolic disorder” is used torefer to disorders which affect how the body processes substances neededto carry out physiological functions, excluding autoimmune diabetes.

The invention provides methods for inhibiting TNFα activity in a subjectsuffering from such a metabolic disorder, which method comprisesadministering to the subject an antibody, antibody portion, such thatTNFα r activity in the subject suffering from a metabolic disorder isinhibited. TNFα antibodies can also be used to treat subjects who are atrisk of developing a metabolic disorder.

Metabolic disorders are often associated with arthritis, includingrheumatoid arthritis. In one embodiment, a TNFα inhibitor, such as anantibody, is used in a multiple-variable dose regimen in a subject whosuffers from a metabolic disorder associated with rheumatoid arthritis.In another embodiment, the invention comprises administering a TNFαantibody to treat disorders associated with diabetes or obesity.

Examples of animal models for evaluating the efficacy of a TNFα antibodyfor the treatment of a metabolic disorder include NOD transgenic mice,Akita mice, NSY transgenic mice and ob/ob mice (see Baeder et al. (1992)Clin Exp Immunol. 89:174; Haseyama et al. (2002) Tohoku J Exp Med.198:233; Makino et al. (1980): Exp. Anim. 29:1; Kolb (1987)Diabetes/Metabolism Reviews 3:751; Hamada et al. (2001) Metabolism.50:1282; Coleman, (1978) Diabeiologia, 14:141; Bailey et al. (1982) Int.J. Obesity 6:11). Examples of animal models used to study vasculitisincludes the mouse HSV model (Behcet's disease), the mouse L. caseimodel (Kawasaki's disease), and the mouse ANCA model (Kawasaki'sdisease). Other models of vasculitis include the McH5-lpr/lpr strain(Nose et al. (1996) Am. J. Path. 149:1763) and the SCG/Kj strain of mice(Kinjoh et a. (1993) Proc. Natl. Acad Sci., USA 90:3413). These micestrains spontaneously develop crescentic glomerulonephritis andnecrotizing vasculitis of the small arteries and arterioles of thespleen, stomach, heart, uterus and ovaries. These animals develophypergammaglobulinemia and ANCA autoantibodies that react withmyeloperoxidase (MPO). Additionally, immunization of rats with human MPOresults in ANCA-associated necrotizing crescentic glomerulonephritis(Brouwer et al. (1993) J. Exp. Med. 177:905).

Metabolic disorders affect how the body processes substances needed tocarry out physiological functions. A number of metabolic disorders ofthe invention share certain characteristics, i.e. they are associatedthe insulin resistance, lack of ability to regulate blood sugar, weightgain, and increase in body mass index. Examples of metabolic disordersinclude diabetes and obesity. Examples of diabetes include type 1diabetes mellitus, type 2 diabetes mellitus, diabetic neuropathy,peripheral neuropathy, diabetic retinopathy, diabetic ulcerations,retinopathy ulcerations, diabetic macrovasculopathy, and obesity.Examples of metabolic disorders which can be treated usingmultiple-variable dose methods comprising administration of a TNlPantibody are described in more detail below:

1. Diabetes

Tumor necrosis factor has been implicated in the pathophysiology ofdiabetes. (see e.g., Navarro et al. (2003) Am J Kidney Dis. 42:53;Daimon et al. (2003) Diabetes care. 26:2015; Zhang et al. (1999) JTongji Med Univ. 19:203; Barbieri er al. (2003) Am J Hypertens. 16:537)For example, TNFα is implicated in the pathophysiology for insulinresistance. It has been found that serum TNF levels in patients withgastrointestinal cancer correlates with insulin resistance (see e.g.,McCall et al. (1992) Br. J. Surg. 79:1361).

The term “diabetes” or “diabetic disorder” or “diabetes mellitus,” asused interchangeably herein, refers to a disease which is marked byelevated levels of sugar (glucose) in the blood. Diabetes can be causedby too little insulin (a chemical produced by the pancreas to regulateblood sugar), resistance to insulin, or both. Diabetes includes the twomost common types of the disorder, namely type I diabetes and type IIdiabetes, which both result from the body's inability to regulateinsulin. Insulin is a hormone released by the pancreas in response toincreased levels of blood sugar (glucose) in the blood.

The term “type 1 diabetes,” as used herein, refers to a chronic diseasethat occurs when the pancreas produces too little insulin to regulateblood sugar levels appropriately. Type 1 diabetes is also referred to asinsulin-dependent diabetes mellitus. IDMM, juvenile onset diabetes, anddiabetes—type I. Type 1 diabetes represents is the result of aprogressive autoimmune destruction of the pancreatic β-cells withsubsequent insulin deficiency.

The term “type 2 diabetes,” refers to a chronic disease that occurs whenthe pancreas does not make enough insulin to keep blood glucose levelsnormal, often because the body does not respond well to the insulin.Type 2 diabetes is also referred to as noninsulin-dependent diabetesmellitus, NDDM, and diabetes—type II

Diabetes is can be diagnosed by the administration of a glucosetolerance test. Clinically, diabetes is often divided into several basiccategories. Primary examples these categories include, autoimmunediabetes mellitus, non-insulin-dependent diabetes mellitus (type 1NDDM), insulin-dependant diabetes mellitus (type 2 IDDM), non-autoimmunediabetes mellitus, non-insulin-dependant diabetes mellitus (type 2NIDDM), and maturity-onset diabetes of the young (MODY). A furthercategory, often referred to as secondary, refers to diabetes broughtabout by some identifiable condition which causes or allows a diabeticsyndrome to develop. Examples of secondary categories include, diabetescaused by pancreatic disease, hormonal abnormalities, drug- orchemical-induced diabetes, diabetes caused by insulin receptorabnormalities, diabetes associated with genetic syndromes, and diabetesof other causes. (see e.g., Harrison's (1996) 14^(th) ed., New York,McGraw-Hill).

Diabetes is often treated with diet, insulin dosages, and variousmedications described herein. Accordingly, a TNFα antibody may also beadministered in combination with agents commonly used to treat metabolicdisorders and pain commonly associated with diabetes.

In addition, the phrase “disorders associated with diabetes,” as usedherein, refers to conditions and other diseases which are commonlyassociated with or related to diabetes. Example of disorders associatedwith diabetes include, for example, hyperglycemia, hyperinsulinemia,hyperlipidaemia, insulin resistance, impaired glucose metabolism,obesity, diabetic retinopathy, macular degeneration, cataracts, diabeticnephropathy, glomerulosclerosis, diabetic neuropathy, erectiledysfunction, premenstrual syndrome, vascular restenosis, ulcerativecolitis, coronary heart disease, hypertension, angina pectoris,myocardial infarction, stroke, skin and connective tissue disorders,foot ulcerations, metabolic acidosis, arthritis, and osteoporosis.

Diabetes manifests itself in the foregoing categories and can causeseveral complications that are discussed in the following sections.Accordingly, the antibody, or antigen-binding fragment thereof, of theinvention can be used to treat diabetes. In one embodiment, a TNFα eantibody, or antigen-binding fragment thereof, is used to treat diabetesassociated with the above identified categories. In another embodiment,the invention includes administering a TNFα antibody to treat disordersassociated with diabetes. Diabetes manifests itself in manycomplications and conditions associated with diabetes, including thefollowing categories:

a. Diabetic Neuropathy and Peripheral Neuropathy

Tumor necrosis factor has been implicated in the pathophysiology ofdiabetic neuropathy and peripheral neuropathy. (See Benjafield et al.(2001) Diabetes Care. 24:753; Qiang et al. (1998) Diabetologia. 41:1321;Pfeiffer et al. (1997) Horm Metab Res. 29:111).

The term “neuropathy,” also referred to as nerve darnage-diabetic, asused herein, refers to a common complication of diabetes in which nervesare damaged as a result of hyperglycemia (high blood sugar levels). Avariety of diabetic neuropathies are recognized, such as distalsensorimotror polyneuropathy, focal motor neuropathy, and autonomicneuropathy.

The term “peripheral neuropathy,” also known as peripheral neuritis anddiabetic neuropathy, as used herein, refers to the failure of the nervesto carry information to and from the brain and spinal cord. Peripheralneuropathy produces symptoms such as pain, loss of sensation, and theinability to control muscles. In some cases, the failure of nerves tocontrol blood vessels, intestinal function, and other organs results inabnormal blood pressure, digestion, and loss of other basic involuntaryprocesses. Peripheral neuropathy may involve damage to a single nerve ornerve group (mononeuropathy) or may affect multiple nerves(polyneuropathy).

Neuropathies that affect small myelinated and unmyelinated fibers of thesympathetic and parasympathetic nerves are known as “peripheralneuropathies.” Furthermore, the related disorder of peripheralneuropathy, also known as peripheral neuritis and diabetic neuropathy,refers to the failure of the nerves to carry information to and from thebrain and spinal cord. This produces symptoms such as pain, loss ofsensation, and the inability to control muscles. In some cases, failureof nerves controlling blood vessels, intestinal function, and otherorgans results in abnormal blood pressure, digestion, and loss of otherbasic involuntary processes. Peripheral neuropathy may involve damage toa single nerve or nerve group (mononeuropathy) or may affect multiplenerves (polyneuropathy).

The term “diabetic neuropathy” refers to a common complication ofdiabetes in which nerves are damaged as a result of hyperglycemia (highblood sugar levels). Diabetic neuropathy is also referred to asneuropathy and nerve damage-diabetic. A variety of diabetic neuropathiesare recognized, such as distal sensorimotror polyneuropathy, focal motorneuropathy, and autonomic neuropathy.

b. Diabetic Retinopathy

Tumor necrosis factor has been implicated in the pathophysiology ofdiabetic retinopathy (Scholz et al. (2003) Trends Microbiol. 11:171).The term “diabetic retinopathy” as used herein, refers to progressivedamage to the eye's retina caused by long-term diabetes. Diabeticretinopathy, includes proliferative retinopathy.

Proliferative neuropathy in turn includes neovascularization, pertinalhemmorrhave anti retinal detachment.

In advanced retinopathy, small vessels proliferate on the surface of theretina. These blood vessels are fragile, tend to bleed and can causeperetinal hemorrhages. The hemorrhage can obscure vision, and as thehemorrhage is resorbed fibrous tissue forms predisposing to retinaldetachments and loss of vision. In addition, diabetic retinopathyincludes proliferative retinopathy which includes neovascularization,pertinal hemmorrhave and retinal detachment. Diabetic retinopathy alsoincludes “background retinopathy” which involves changes occurring withthe layers of the retina.

c. Diabetic Ulcerations and Retinopathy Ulcerations

Tumor necrosis factor has been implicated in the pathophysiology ofdiabetic ulcerations, (see Lee et al. (2003) Hum Immunol. 64:614;Navarro et al. (2003) Am J Kidney Dis. 42:53; Daimon et al (2003)Diabetes Care. 26:2015; Zhang et al. (1999) J Tongji Med Univ. 19:203;Barbieri et al. (2003) Am J Hypertens. 16:537; Venn et al. (1993)Arthritis Rheum. 36:819; Westacott et al. (1994) J Rheumwnatol.21:1710).

The term “diabetic ulcerations,” as used herein, refers to an ulcerwhich results as a complication of diabetes. An ulcer is a crater-likelesion on the skin or mucous membrane caused by an inflammatory,infectious, malignant condition, or metabolic disorder. Typicallydiabetic ulcers can be found on limbs and extremities, more typicallythe feet. These ulcers, caused by diabetic conditions, such asneuropathy and a vacuair insuffciency, can lead to ischemia and poorwound healing. More extensive ulcerations may progress to osteomyelitis.Once osteomyelitis develops, it may be difficult to eradicate withantibiotics alone and amputation maybe necessary.

The term “retinopathy ulcerations,” as used herein refers to an ulcerwhich causes or results in damages to the eye and the eye's retina.Retinopathy ulcerations may include conditions such has retinopathichemorrhages.

d. Diabetic Macrovasculopathy

Tumor necrosis factor has been implicated in the pathophysiology ofdiabetic macrovasculopathy (Devaraj et al. (2000) Circulation. 102:191;Hattori et al. (2000) Cardiovasc Res. 46:188; Clausell et al. (1999)Cardiovasc Pathol. 8:145). The term “diabetic macrovasculopathy,” alsoreferred to as “macrovascular disease,” as used herein, refers to adisease of the blood vessels that results from diabetes. Diabeticmacrovasculopathy complication occurs when, for example, fat and bloodclots build up in the large blood vessels and stick to the vessel walls.Diabetic macrovasculopathies include diseases such as coronary disease,cerebrovascular disease, and peripheral vascular disease, hyperglycaemiaand cardiovascular disease, and strokes.

2. Obesity

Tumor necrosis factor has been implicated in the pathophysiology ofobesity (see e.g., Pihlajamaki J et al. (2003) Obes Res. 11:912;Barbieri et al. (2003) Am J Hypertens. 16:537; Tsuda et al. (2003) JNutr. 133:2125). The term “obesity” as used herein, refers to acondition in which the subject has an excess of body fat relative tolean body mass. In one embodiment, obesity refers to a condition inwhich an individual weighs at least about 20% or more over the maximumdesirable for their height. When an adult is more than 100 poundsoverweight, he or she is considered to be “morbidly obese.” In anotherembodiment, obesity is defined as a BMI (body mass index) over 30 kg/m2.Obesity increases a person's risk of illness and death due to diabetes,stroke, coronary artery disease, hypertension, high cholesterol, andkidney and gallbladder disorders. Obesity may also increase the risk forsome types of cancer, and may be a risk factor for the development ofosteoarthritis and sleep apnea.

K. Anemia

TNFα has been implicated in the pathophysiology of a wide variety ofanemias (see e.g., Jongen-Lavrencie et al. (1997) J. Rheumatol. 24:1504;Demeter et al. (2002) Ann Hematol. 81:566; DiCato (2003) The Oncologist8 (suppl 1):19). The invention provides a method for inhibiting TNFαactivity in a subject suffering from anemia, which method comprisesadministering to the subject an antibody, antibody portion, such thatTNFα activity in the subject suffering from anemia is inhibited. In oneembodiment, the anemia is associated with rheumatoid arthritis.

The term “anemia” as used herein, refers to an abnormally low number ofcirculating red cells or a decreased concentration of hemoglobin in theblood. Examples of anemia related to rheumatoid arthritis include, forexample, anemia of chronic disease, iron deficiency anemia, andautoimmune hemolytic anemia. In one embodiment, the invention provides amethod of treating anemias related to, for example, anemias related torheumatoid arthritis, anemias of infection and chronic inflammatorydiseases, iron deficiency anemia, autoimmune hemolytic anemia,myelophthisic anemia, aplastic anemia, hypoplastic anemia, pure red cellaplasia and anemia associated with renal failure or endocrine disorders,megaloblastic anemias, defects in heme or globin synthesis, anemiacaused by a structural defect in red blood cells, e.g., sickle-cellanemia, and anemias of unknown origins such as sideroblastic anemia,anemia associated with chronic infections such as malaria,trypanosomiasis, HIV, hepatitis virus or other viruses, andmyelophthisic anemias caused by marrow deficiencies.

Examples of animal models used to study anemia include rats inoculatedwith peptidolglycan-polysaccharide polymers (see Coccia et al., (2001)Exp Hematology. 29:1201-1209). Examples of animal models used to studypain are well known in the art, and include the rat sciatic nerveligation model, and the rat segmental spinal nerve ligation model (seeBennett and Zic, (1988) Pain. 33:87-107; Kim and Chang, (1992) Pain50:355-363).

L. Pain

TNFα has been implicated in the pathophysiology of a wide variety ofpain syndromes (see e.g., Sorkin et al. (1997) Neuroscience. 81:255;Huygen et al. (2002) Mediators Inflamm. 11:47; Parada et al. (2003) EurJ Neurosci. 17:1847). The term “pain” as used herein, refers to alltypes of pain. The term shall refer to acute and chronic pains, such asneuropathic pain and post-operative pain, chronic lower back pain,cluster headaches, herpes neuralgia, phantom limb pain, central pain,dental pain, opioid-resistant pain, visceral pain, surgical pain, boneinjury pain, pain during labor and delivery, pain resulting from burns,including sunburn, post partum pain, migraine, angina pain, andgenitourinary tract-related pain including cystitis. The term alsoincludes nociceptive pain or nociception.

The invention provides methods for inhibiting TNFα activity in a subjectsuffering from such a pain disorder, which method comprisesadministering to the subject an antibody, antibody portion, such thatTNFα activity in the subject suffering from pain is inhibited. Pain hasbeen defined in a variety of ways, including nociceptive pain andneuropathic pain. The most commonly experienced form of pain may bedefined as the effect of a stimulus on nerve endings, which results inthe transmission of impulses to the cerebrum. Pain is also commonlyassociated with inflammatory disorders, including, for example,rheumatoid arthritis. In one embodiment, the antibody of the inventionis used to treat a subject who suffers from pain associated withrheumatoid arthritis. Examples of pain disorders in which TNFα activityis detrimental are discussed further below.

1. Neuropathic Pain

Tumor necrosis factor has been implicated in the pathophysiology ofneuropathic pain (see Sommer (1999) Schmerz. 13:315; Empl et al., (2001)Neurology. 56:1371; Schafers et al. (2003) J Neurosci. 23:3028). As usedherein the term “neuropathic pain” refers to pain that results frominjury to a nerve, spinal cord, or brain, and often involves neuralsupersensitivity. Examples of neuropathic pain include chronic lowerback pain, pain associated with arthritis, cancer-associated pain,herpes neuralgia, phantom limb pain, central pain, opioid resistantneuropathic pain, bone injury pain, and pain during labor and delivery.Other examples of neuropathic pain include post-operative pain, clusterheadaches, dental pain, surgical pain, pain resulting from severe, forexample third degree, burns, post partum pain, angina pain,genitourinary tract related pain, and including cystitis.

Neuropathic pain is distinguished from nociceptive pain. Pain involvinga nociceptive mechanism usually is limited in duration to the period oftissue repair and generally is alleviated by available analgesic agentsor opioids (Myers (1995) Regional Anesthesia 20:173). Neuropathic paintypically is long-lasting or chronic and often develops days or monthsfollowing an initial acute tissue injury. Neuropathic pain can involvepersistent, spontaneous pain as well as allodynia, which is a painfulresponse to a stimulus that normally is not painful. Neuropathic painalso can be characterized by hyperalgesia, in which there is anaccentuated response to a painful stimulus that usually is trivial, suchas a pin prick. Unlike nociceptive pain, neuropathic pain generally isresistant to opioid therapy (Myers, supra, 1995). Accordingly,antibodies obtained using methods of the invention can be used to treatneuropathic pain.

2. Nociceptive Pain

As used herein the term “nociceptive pain” refers to pain that istransmitted across intact neuronal pathways, i.e., pain caused by injuryto the body. Nociceptive pain includes somatic sensation and normalfunction of pain, and informs the subject of impending tissue damage.The nociceptive pathway exists for protection of the subject. e.g., thepain experienced in response to a burn). Nociceptive pain includes bonepain, visceral pain, and pain associated with soft tissue.

Tumor necrosis factor has been implicated in the pathophysiology ofvisceral pain (see Coelho et al. (2000) Am J Physiol Gastrointest LiverPhysiol. 279:G781; Coelho et al. (2000) Brain Res Bull. 52:223).Visceral pain is used to refer to nociceptive pain that is mediated byreceptors on A-delta and C nerve fibers. A-delta and C-nerve fibers arewhich are located in skin, bone, connective tissue, muscle and viscera.Visceral pain can be vague in distribution, spasmodic in nature and isusually described as deep, aching, squeezing and colicky in nature.Examples of visceral pain include pain associated with a heart attack,wherein the visceral pain can be felt in the arm, neck and/or back, andliver capsule pain, wherein the visceral pain can be felt in the backand/or right shoulder. Accordingly, antibodies obtained using theinvention can be used to treat visceral pain.

M. Hepatic Disorders

TNFα has been implicated in the pathophysiology of a wide variety ofhepatic disorders (see e.g., Colletti et al. (1990) J Clin Invest.85:1936; Tiegs (1997) Acta Gastroenterol Belg. 60:176; Fernandez et al.(2000) J Endotoxin Res. 6:321). The invention provides methods forinhibiting TNFα activity in a subject suffering from such a hepaticdisorder.

As used herein, the term “a hepatic disorder in which TNFα activity isdetrimental” is intended to include diseases and other disorders of theliver or conditions associated with hepatocellular injury or a biliarytract disorders in which the presence of TNFα in a subject sufferingfront the disorder has been shown to be or is suspected of being eitherresponsible for the pathophysiology of the disorder or a factor thatcontributes to a worsening of the disorder. Accordingly, a hepaticdisorder in which TNFα activity is detrimental is a disorder in whichinhibition of TNFα activity is expected to alleviate the symptoms and/orprogression of the hepatic disorder. In one embodiment, hepaticdisorders refers to a human liver disease or condition associated withhepatocellular injury or a biliary tract disorder excluding hepatitis,alcoholic hepatitis, and viral hepatitis.

Examples of animal models used for evaluating the therapeutic efficacyof an agent for treating a hepatic disorder using multiple-variable dosemethods include the chimpanzee hepatitis C virus model (see Shimizu etal. (1990) Proc Natl Acad Sci. USA 87:6441). Examples of animal modelsused to study skin and nail disorder disorders include, for example, thesevere combined immunodeficient (SCID) mouse model (psoriasis) and theSmith line (SL) chicken and depigmenting mouse (vitiligo) (see Nickoloff(2000) Investig Dermatol Symp Proc. 5:67; Austin et al. (1995) Am JPathol. 146:1529; Lerner et al. (1986) J Invest Dermatol. 87:299).

Hepatic disorders include many diseases and disorders wherein the liverfunctions improperly or ceases to function. Hepatocellular injuries caninclude alcoholic cirrhosis, α1 antitypsin deficiency, autoimmunecirrhosis, cryptogenic cirrhosis, fulminant hepatitis, hepatitis B andC, and steatohepatitis. Examples of biliary tract disorders includecystic fibrosis, primary biliary cirrhosis, sclerosing cholangitis andbiliary obstruction (Wiesner (1996) “Current Indications, ContraIndications and Timing for Liver Transplantation” in Transplantation ofthe Liver, Saunders (publ.); Busuttil and Klintmalm (eds.) Chapter 6;Klein (1998) Partial Hypertension: The Role of Liver Transplantation,Musby (publ.) in Current Surgical Therapy 6.sup.th Ed. Cameron, J. (ed).

The term “hepatitis” refers to inflammation of the liver. Hepatitis canbe caused by infections with various organisms, including bacteria,viruses (Hepatitis A, B, C, etc.), or parasites. Chemical toxins such asalcohol, drugs, or poisonous mushrooms can also damage the liver andcause it to become inflamed. A rare but extremely dangerous cause ofhepatitis results from overdose of acetaminophen (Tylenol), which can bedeadly. In addition, immune cells in the body may attack the liver andcause autoimmune hepatitis. Hepatitis may resolve quickly (acutehepatitis), or cause long-term disease (chronic hepatitis). In someinstances, progressive liver damage or liver failure may result. Theincidence and severity of hepatitis vary depending on many factors,including the cause of the liver damage and any underlying illnesses ina patient.

In one embodiment, the invention features methods for treating a hepaticdisorder in which TNFα activity is detrimental, comprising administeringto a subject an effective amount of a TNFα inhibitor in an inductiondose and subsequently in a treatment dose, such that said disorder istreated. In one embodiment, the hepatic disorder is selected from thegroup consisting of hepatitis C virus, autoimmune hepatitis, fatty-liverdisease, hepatitis B virus, hepatotoxicity, and non-alcoholic hepatitis,including non-alcoholic steatohepatitis (NASH). Examples of hepaticdisorders are further described below.

1. Hepatitis C Virus (HCV)

Tumor necrosis factor has been implicated in the pathophysiology of thehepatitis C virus (see Gonzalez-Amaro. (1994) J Exp Med. 179:841; Nelsonet al. (1997) Dig Dis Si 42:2487; Kallinowski et al. (1998) Clin ExpImmunol. 111:269). The term “hepatitis C virus” or “HCV” is used todescribe the hepatitis virus which is the causative agent of non-A,non-B hepatitis. Hepatitis C virus causes an inflammation of the liver.HCV infection causes hepatitis C. Hepatitis C in the acute stage is, ingeneral, milder than hepatitis B, but a greater proportion of suchinfections become chronic. HCV is a major cause of acute hepatitis andchronic liver disease, including cirrhosis and liver cancer. HCV is oneof the viruses (A, B, C, D, and E), which together account for the vastmajority of cases of viral hepatitis. It is an enveloped RNA virus inthe flaviviridae family which appears to have a narrow host range. Animportant feature of the virus is the relative mutability of its genome,which in turn is probably related to the high propensity (80%) ofinducing chronic infection. HCV is clustered into several distinctgenotypes which may be important in determining the seventy of thedisease and the response to treatment. In one embodiment, the inventionprovides a multiple-variable dose method for treating HCV.

2. Autoimmune Hepatitis (AIH)

Tumor necrosis factor has been implicated in the pathophysiology ofautoimmune hepatitis (see Cookson et al., (1999) Hepatology 30:851;Jazrawi et al., (2003) Liver Transpl. 9:377). As used herein.“autoimmune hepatitis” refers to a hepatic disorder characterized byinflammation of the liver caused by rogue immune cells that mistake theliver's normal cells for a foreign issue or pathogen (disease-causingagent). Autoimmune hepatitis is often responsible for a progressivedestruction of the hepatic parenchyma with a high mortality if leftuntreated (Johnson et al. (1993) Hepatology, 18:998). One of thecharacteristic of autoimmune hepatitis is the presence of circulatingautoantibodies in almost 90% of patients' sera. Such antibodies can beused to identify subjects who have autoimmune hepatitis.

Clinical and serological differences between patients have lead to theclassification of AIH into two types. Type 1 is characterized by thepresence of anti-smooth muscle (SMA) and/or anti-nuclear antibodies(ANA) in patients' sera, while sera from Type II patients showanti-liver kidney microsomal antibodies type 1 (LKM1) (Homberg et al.,(1987) Hepatology, 7:1333; Maggiore et al. (1993) J. Pediatr.Gastroenterol Nutr. 17:376). A serological marker, anti-liver cytosoltype I antibodies (LC1), has been identified in 30% of patients with anAIH type II. In addition, LC1 proved to be the only serological markerin 10% of patients tested (Martini et al. (1988) Hepatology, 8:1662). Inone embodiment, the method of the invention is used to treat AIH.

3. Fatty-Liver Disease

Tumor necrosis factor has been implicated in the pathophysiology offatty-liver disease (see Valenti et al., (2002) Gastroenerology 122:274;Li et al., (2003) Hepatology 37:343). Fatty-liver disease refers to adisease wherein fat (hepatocytes) is excessively accumulated in theliver. Fatty liver disease is believed to be caused by supernutrition,hyperingestion of alcohol, diabetes and side effects due toadministration of pharmaceuticals. Fatty liver disease can cause severediseases such as chronic hepatitis and hepatic cirrhosis. In patientswith fatty liver disease, lipids, particularly neutral fat, accumulatein hepatocytes to the extent that the amount exceeds the physiologicallypermissible range. Front a biochemical point of view, a standard forjudgment of fatty liver is that the weight of neutral fat is about 10%(100 mg/g wet weight) or more of the wet weight of hepatic tissue. Inone embodiment, the method of the invention is used to treat fatty liverdisease.

4. Hepatitis B Virus (HBV)

Tumor necrosis factor has been implicated in the pathophysiology ofhepatitis B virus (see Kasahara et al., (2003) J Virol. 77:2469; Wang(2003) World J Gastroenterol. 9:641; Bierner et al. (2003) J Virol.77:4033). The term “hepatitis B virus” (HBV) is used to describe thevirus (serum hepatitis virus) which produces viral hepatitis type B inhumans. This is a viral disease with a long incubation period (about 50to 160 days) in contrast to hepatitis A virus (infectious hepatitisvirus) which has a short incubation period. The hepatitis B virus isusually transmitted by injection of infected blood or blood derivativesor merely by use of contaminated needles, lancets or other instruments.Clinically and pathologically, the disease is similar to viral hepatitistype A; however, there is no cross-protective immunity. Viral antigen(HBAg) is found in the serum after infection.

Hepatitis B virus infects humans at a very high rate. Most people whobecome infected with Hepatitis B get rid of the virus within 6 months,wherein a short infection is known as an “acute” case of Hepatitis B. Itis estimated that at least about 300 million people are chronic carriersof HBV. Infection with the virus results in a range of clinical symptomsincluding minor flu-like symptoms to death. In one embodiment, themultiple-variable dose method of the invention is used to treat HBVinfection.

5. Hepatotoxicity

Tumor necrosis factor has been implicated in the pathophysiology ofhepatotoxicity (see Bruceoleri et al. (1997) Hepatology 25:133; Lusteret al. (2000) Ann NY Acad Sci. 919:214; Simeonova et al. (2001) ToxicolAppl Pharmacol. 177:112). The term hepatotoxicity refers to liver damagecaused by medications and other chemicals or drugs. The best indicatorfor identifying liver toxicity in a subject is the elevation of certainenzyme measurements in the blood, such as AST (aspartateaminotransferase), ALT (alanine aminotransferase), and GOT (glutamateoxalacetate transaminase).

Hepatotoxicity can cause permanent injury and death. Initial symptoms ofhepatotoxicity can include acute gastrointestinal symptoms, e.g., severediarrhea. The second phase of hepatotoxicity is characterized byabatement of symptoms. During this apparent subsidence, biochemicalevidence of hepatic injury appears. Oliguria (decreased urine output) isusual during the second phase. The third phase, that of overt hepaticdamage, becomes clinically apparent 3 to 5 days after ingestion of thechemical, with the appearance of jaundice. Renal failure may also occur.The symptoms of chemically-induced (drug-induced) hepatitis are similarto that of infectious hepatitis. In one embodiment, the method of theinvention is used to treat hepatotoxicity.

6. Liver Failure (e.g. Chronic Liver Failure)

Tumor necrosis factor has been implicated in the pathophysiology ofliver failure (e.g. chronic liver failure) (see Takenaka et al., (1998)Dig Dis Sci. 43:887; Nagaki et al. (1999) J Hepatol. 31:997; Streetz etal., (2000) Gasroenterology. 119:446. Liver failure, including chronicliver failure, usually develops over a period of years and is caused bya repeated insult to the liver (such as alcohol abuse or infection withhepatitis virus) which slowly damages the organ. Less commonly, liverfailure is acute, and occurs over a period of days or weeks. Causes ofacute liver failure include hepatitis virus infections, drugs,pregnancy, autoimmune disease, and sudden low blood flow to the liver.In one embodiment, the method of the invention is used to treat liverfailure.

7. Non-Alcoholic Hepatitis, Including NASH

Tumor necrosis factor has been implicated in the pathophysiology ofnon-alcoholic hepatitis, including nonalcoholic steatohepatitis (seeCrespo et al., (2001) Hepatology. 34:1158; Pessayre et al. (2002)282(2):G193). The term “nonalcoholic steatohepatitis” or “NASH” refersto the development of histologic changes in the hlver that arecomparable to those induced by excessive alcohol intake, but in theabsence of alcohol abuse. NASH is characterized by macrovesicular and/ormicrovesicular steatosis, lobular and portal inflammation, andoccasionally Mallory bodies with fibrosis and cirrhosis. NASH is alsocommonly associated with hyperlipidemia, obesity, and type II diabetesmellitus.

Additional clinical conditions which characterize hepatic steatosis andinflammation include excessive fasting, jejunoileal bypass, totalparental nutrition, chronic hepatitis C, Wilson's disease, and adversedrug effects such as those from corticosteroids, calcium channelblockers, high dose synthetic estrogens, methotrexate and amiodarone.Thus, the term “nonalcoholic steatohepatitis” can be used to describethose patients who exhibit these biopsy findings, coupled with theabsence of (a) significant alcohol consumption, (b) previous surgery forweight loss, (c) history of drug use associated with steatohepatitis,(d) evidence of genetic liver disease or (e) chronic hepatitis Cinfection (see. e.g., Ludwig et al., (1980) Mayo Clin. Proc. 55:434;Powell et al. (1990) Hepatol. 11:74). In one embodiment, the antibodiesobtained using the method of the invention are used to treat NASH.

N. Skin and Nail Disorders

Tumor necrosis factor has been implicated in the pathophysiology of skinand nail disorders. In one embodiment, antibodies obtained using themethod of the invention are administered to treat skin and naildisorders. The term “skin disorder” or “skin disease” as usedinterchangeably herein, refers to abnormalities, other than injurywounds, of the skin which have induced a state of inflammation. In oneembodiment, the skin disorder of the invention is an inflammatory skindisorder, wherein the skin is characterized by capillary dilatation,leukocytic infiltration, redness, heat, and/or pain. Examples of skindisorders include, but are not limited to, psoriasis, pemphigusvulgaris, scleroderma, atopic dermatitis, sarcoidosis, erythema nodosum,hidradenitis suppurative, lichen planus, Sweet's syndrome, and vitiligo.As used herein, the term “skin and nail disorder in which TNFα activityis detrimental” is intended to include skin and/or nail disorders andother disorders in which the presence of TNFα in a subject sufferingfrom the disorder has been shown to be or is suspected of being eitherresponsible fir the pathophysiology of the disorder of a factor thatcontributes to a worsening of the disorder, e.g., psoriasis.Accordingly, skin and nail disorders in which TNFα activity isdetrimental are disorders in which inhibition of TNFα activity isexpected to alleviate, the symptoms and/or progression of the disorder.The use of the antibodies, antibody portions, and other TNFα inhibitorsof the invention in the treatment of specific skin and nail disorders isdiscussed further below. In certain embodiments, the treatment method ofthe invention is performed in combination with another therapeuticagent, as described below. In one embodiment, the antibodies obtainedusing the method of the invention comprising administering a TNFantibody in combination with another therapeutic agent is used for thetreatment of psoriasis and the treatment of psoriasis associated witharthritis.

1. Psoriasis

Tumor necrosis factor has been implicated in the pathophysiology ofpsoriasis (Takematsu et al. (1989) Arch Dermatol Res. 281:398; Victorand Gottlieb (2002) J Drugs Dermatol. 1:264). The term “psoriasis” asused herein, refers to skin disorders associated with epidermalhyperplasia. Example of psoriasis include, but are not limited to,chronic plaque psoriasis, guttate psoriasis, inverse psoriasis, postularpsoriasis, psoriasis vulgaris, and erythrodermic psoriasis. Psoriasiscan also be associated with other inflammatory disorders, includinginflammatory bowel disease (IBD) and rheumatoid arthritis (RA).

Psoriasis is described as a skin inflammation (irritation and redness)characterized by frequent episodes of redness, itching, and thick, dry,silvery scales on the skin. In particular, lesions are formed whichinvolve primary and secondary alterations in epidernial proliferation,inflammatory responses of the skin, and an expression of regulatorymolecules such as lymphokines and inflammatory factors. Psoriatic skinis morphologically characterized by an increased turnover of epidermalcells, thickened epidermis, abnormal keratinization, inflammatory cellinfiltrates into the epidermis and polymorphonuclear leukocyte andlymphocyte infiltration into the epidermis layer resulting in anincrease in the basal cell cycle. Psoriasis often involves the nails,which frequently exhibit pitting, separation of the nail, thickening,and discoloration. Psoriasis is often associated with other inflammatorydisorders, for example arthritis, including rheumatoid arthritis,inflammatory bowel disease (IBD), and Crohn's disease. Approximately onethird of subjects with psoriasis also have psoriatic arthritis (PsA)which, as described above, causes stiffness, swelling of the joints,pain, and reduced range of motion (Greaves et al. (1995) N. Eng. J. Med.332:581).

Evidence of psoriasis is most commonly seen on the trunk, elbows, knees,scalp, skin folds, or fingernails, but it may affect any or all parts ofthe skin. Normally, it takes about a month for new skin cells to move upfrom the lower layers to the surface. In psoriasis, this process takesonly a few days, resulting in a build-up of dead skin cells andformation of thick scales. Symptoms of psoriasis include: skin patches,that are dry or red, covered with silvery scales, raised patches ofskin, accompanied by red borders, that may crack and become painful, andthat are usually located on the elbows, knees, trunk, scalp, and hands;skin lesions, including pustules, cracking of the skin, and skinredness; joint pain or aching which may be associated with of arthritis,e.g., psoriatic arthritis.

Treatment for psoriasis often includes a topical corticosteroids,vitamin D analogs, and topical or oral retinoids, or combinationsthereof. In one embodiment, the TNFα antibody of the invention isadministered in combination with or the presence of one of these commontreatments. Additional therapeutic agents which can be combined with theTNFα antibody obtained using the methods of the invention for treatmentof psoriasis are described in more detail below.

The diagnosis of psoriasis is usually based on the appearance of theskin. Additionally a skin biopsy, or scraping and culture of skinpatches may be needed to rule out other skin disorders. An x-ray may beused to check for psoriatic arthritis if joint pain is present andpersistent.

Improvements in psoriasis in a subject can be monitored by the subject'sPsoriasis Area and Severity Index Score (PASI). The method fordetermining the PASI has been described in Fredriksson and Pettersson(1978) Dermatologica 157:238 and Marks et al. (1989) Arch Dermatol125:235. Briefly, the index is based on evaluation of four anatomicsites, including the head, upper extremities, trunk, and lowerextremities, for erythema, induration, and desquamation using a 5 pointscale (0=no symptoms; 1=slight; 2=moderate; 3=marked; 4=very marked).Based on the extent of lesions in a given anatomic site, the areaaffected is assigned a numerical value (0=0; 1=<10%: 2=10-29%; 3=30-49%;4=50-69%; 5=70=89%; 6=90-100%). The PASI score is then calculated,wherein the possible range of PASI score is 0.0 to 72.0 with the highestscore representing complete erythroderma of the severest degree.

In one embodiment of the invention, a TNFα antibody is used for thetreatment of psoriasis, including chronic plaque psoriasis, guttatepsoriasis, inverse psoriasis, postular psoriasis, pemphigus vulgaris,erythrodermic psoriasis, psoriasis associated with inflammatory boweldisease (IBD), and psoriasis associated with rheumatoid arthritis (RA).In another embodiment, a TNFα antibody, such as adalimumab, is used totreat subjects who have psoriasis in combination with PsA. Specifictypes of psoriasis included in the treatment methods of the inventionare described in detail below:

a. Chronic Plaque Psoriasis

Tumor necrosis factor has been implicated in the pathophysiology ofchronic plaque psoriasis (Asadullah et al. (1999) Br J Dermatol.141:94). Chronic plaque psoriasis (also referred to as psoriasisvulgaris) is the mot common form of psoriasis. Chronic plaque psoriasisis characterized by raised reddened patches of skin, ranging fromcoin-sized to much larger. In chronic plaque psoriasis, the plaques maybe single or multiple, they may vary in size from a few millimeters toseveral centimeters. The plaques are usually red with a scaly surface,and reflect light when gently scratched, creating a “silvery” effect.Lesions (which are often symmetrical) from chronic plaque psoriasisoccur all over body, but with predilection for extensor surfaces,including the knees, elbows, lumbosacral regions, scalp, and nails.Occasionally chronic plaque psoriasis can occur on the penis, vulva andflexures, but scaling is usually absent. Diagnosis of patients withchronic plaque psoriasis is usually based on the clinical featuresdescribed above. In particular, the distribution, color and typicalsilvery scaling of the lesion in chronic plaque psoriasis arecharacteristic of chronic plaque psoriasis.

b. Guttate psoriasis

Guttate psoriasis refers to a form of psoriasis with characteristicwater drop shaped scaly plaques. Flares of guttate psoriasis generallyfollow an infection, most notably a streptococcal throat infection.Diagnosis of guttate psoriasis is usually based on the appearance of theskin, and the tact that there is often a history of recent sore throat.

c. Inverse Psoriasis

Inverse psoriasis is a form of psoriasis in which the patient hassmooth, usually moist areas of skin that are red and inflammed, which isunlike the scaling associated with plaque psoriasis. Inverse psoriasisis also referred to as interiginous psoriasis or flexural psoriasis.Inverse psoriasis occurs mostly in the armpits, groin, under the breastsand in other skin folds around the genitals and buttocks, and, as aresult of the locations of presentation, rubbing and sweating canirriate the affected areas.

d. Pustular psoriasis

Pustular psoriasis, also referred to as palmer plantar psoriasis, is aform of psoriasis that causes pus-filled blisters that vary in size andlocation, but often occur on the hands and feet. The blisters may belocalized, or spread over large areas of the body. Pustular psoriasiscan be both tender and painful, can cause fevers.

e. Other Psoriasis Disorders

Other examples of psoriatic disorders which can be treated with the TNFαantibody obtained using the methods of the invention includeerythrodemic psoriasis, vulgaris, psoriasis associated with IBD, andpsoriasis associated with arthritis, including rheumatoid arthritis.

2. Pemphigus vulgaris

Pemphigus vulgaris is a serious autoimmune systemic dermatologic diseasethat often affects the oral mucous membrane and skin. The pathogenesisof pemphigus vulgaris is thought to be an autoimmune process that isdirected at skin and oral mucous membrane desmosomes. Consequentially,cells do not adhere to each other. The disorder manifest as largefluid-filled, rupture-prone bullae, and has a distinctive histologicappearance. Anti-inflammatory agents are the only effective therapy forthis disease which has a high mortality rate. Complications that arisein patients suffering from pemphigus vulgaris are intractable pain,interference with nutrition and fluid loss, and infections.

3. Atopic Dermatitis/Eczema

Atopic dermatitis (also referred to as eczema) is a chronic skindisorder categorized by scaly and itching plaques. People with eczemaoften have a family history of allergic conditions like asthma, hayfever, or eczema. Atopic dermatitis is a hypersensitivity reaction(similar to an allergy) which occurs in the skin, causing chronicinflammation. The inflammation causes the skin to become itchy andscaly. Chronic irritation and scratching can cause the skin to thickenand become leathery-textured. Exposure to environmental irritants canworsen symptoms, as can dryness of the skin, exposure to water,temperature changes, and stress.

Subjects with atopic dermatitis can be identified by certain symptoms,which often include intense itching, blisters with oozing and crusting,skin redness or inflammation around the blisters, rash, dry, leatheryskin areas, raw areas of the skin from scratching, and eardischarges/bleeding.

4. Sarcoidosis

Sarcoidosis is a disease in which granulomatous inflammation occurs inthe lymph nodes, lungs, liver, eyes, skin, and/or other tissues.Sarcoidosis includes cutaneous sarcoidosis (sarcoidosis of the skin) andnodular sarcoidosis (sarcoidosis of the lymph nodes). Patients withsarcoidosis can be identified by the symptoms, which often includegeneral discomfort, uneasiness, or an ill feeling; fever; skin lesions.

5. Erythema Nodosum

Erythema nodosum refers to an inflammatory disorder that ischaracterized by tender, red nodules under the skin, typically on theanterior lower legs. Lesions associated with erythema nodosum oftenbegin as flat, but firm, hot red painful lumps (approximately an inchacross). Within a few days the lesions may become purplish, and thenover several weeks fade to a brownish flat patch.

In some instances, erythema nodosum may be associated with infectionsincluding, streptococcus, coccidioidomycosis, tuberculosis, hepatitis B,syphilis, cat scratch disease, tularemia, yersinia, leptospirosispsittacosis, histoplasmosis, mononucleosis (EBV). In other instances,crythema nodosum may be associated with sensitivity to certainmedications including, oral contraceptives, penicillin, sulfonamides,sulfones, barbiturates, hydantoin, phenacetin, salicylates, iodides, andprogestin. Erythema nodosum is often associated with other disordersincluding, leukemia, sarcoidosis, rheumatic fever, and ulcerativecolitis.

Symptoms of erythema nodosum usually present themselves on the shins,but lesions may also occur on other areas of the body, including thebuttocks, calves, ankles, thighs and upper extremities. Other symptomsin subjects with erythema nodosum can include fever and malaise.

6. Hidradenitis Suppurative

Hidradenitis suppurativa refers to a skin disorder in which swollen,painful, inflamed lesions or lumps develop in the groin and sometimesunder the arms and under the breasts. Hidradenitis suppurativa occurswhen apoerine gland outlets become blocked by perspiration or are unableto drain normally because of incomplete gland development. Secretionstrapped in the glands force perspiration and bacteria into surroundingtissue, causing subcutaneous induration, inflammation, and infliction.Hidradenitis suppurativa is confined to areas of the body that containapoerine glands. These areas are the axillae, areola of the nipple,groin, perineum, circumanal, and periumbilical regions.

7. Lichen Planus

Tumor necrosis factor has been implicated in the pathophysiology oflichen planus (Sklayounou et al. (2000) J Oral Pathol Med. 29:370).Lichen planus refers to a disorder of the skin and the mucous membranesresulting in inflammation, itching, and distinctive skin lesions. Lichenplanus may be associated with hepatitis C or certain medications.

8. Sweet's Syndrome

Inflammatory cytokines, including tumor necrosis factor, have beenimplicated in the pathophysiology of Sweet's syndrome (Reuss-Borst etal. (1993) Br J Haematol. 84:356). Sweet's syndrome, which was describedby R. D. Sweet in 1964, is characterized by the sudden onset of fever,leukocytosis, and cutaneous eruption. The eruption consists of tender,erythematous, well-demarcated papules and plaques which show denseneutrophilic infiltrates microscopically. The lesions may appearanywhere, but favor the upper body including the face. The individuallesions are often described as pseudovesicular or pseudopustular, butmay be frankly pustular, bullous, or ulcerative. Oral and eyeinvolvement (conjunctivitis or episcleritis) have also been frequentlyreported in patients with Sweet's syndrome. Leukemia has also beenassociated with Sweet's syndrome.

9. Vitiligo

Vitiligo refers to a skin condition in which there is loss of pigmentfrom areas of skin resulting in irregular white patches with normal skintexture. Lesions characteristic of vitiligo appear as flat depigmentedareas. The edges of the lesions are sharply defined but irregular.Frequently affected areas in subjects with vitiligo include the face,elbo ws and knees, hands and feet, and genitalia.

10. Scleroderma

Tumor necrosis factor has been implicated in the pathophysiology ofscleroderma (Tutuneu et al. (2002) Clin Exp Rheumatol. 20(6 Suppl28):S146; Mackiewiez et al. (2003) Clin Exp Rheumatol. 21:41; Murota etal. (2003) Arthritis Rheum. 48:1117). Scleroderma refers to a diffuseconnective tissue disease characterized by changes in the skin, bloodvessels, skeletal muscles, and internal organs. Sclerodermia is alsoreferred to as CREST syndrome or progressive systemic sclerosis, andusually affects people between the ages 30-50. Women are affected moreoften than men.

The cause of scleroderma is unknown. The disease may produce local orsystemic symptoms. The course and severity of the disease varies widelyin those affected. Excess collagen deposits in the skin and other organsproduce the symptoms. Damage to small blood vessels within the skin andaffected organs also occurs. In the skin, ulceration, calcification, andchanges in pigmentation may occur. Systemic features may includefibrosis and degeneration of the heart, lungs, kidneys andgastrointestinal tract.

Patients suffering from scleroderma exhibit certain clinical features,including, blanching, blueness, or redness of fingers and toes inresponse to heat and cold (Raynaud's phenomenon), pain, stiffness, andswelling of fingers and joints, skin thickening and shiny hands andforearm, esophageal reflux or heartburn, difficulty swallowing, andshortness of breath. Other clinical symptoms used to diagnosescleroderma include, an elevated erythrocyte sedimentation rate (ESR),an elevated rheumatoid factor (RF), a positive antinuclear antibodytest, urinalysis that shows protein and microscopic blood, a chest X-raythat may show fibrosis, and pulmonary function studies that showrestrictive lung disease.

11. Nail Disorders

Nail disorders include any abnormality of the nail. The term “naildisorder” or “nail disease” as used herein, refers to conditions whereinthe fingernails or toenails to abnormal color, shape, texture, orthickness. Specific nail disorders include, but are not limited to,pitting, kollonychia, Beau's lines, spoon nails, onycholysis, yellownails, pterygium (seen in lichen planus), and leukonychia. Pitting ischaracterised by the presence of small depressions on the nail surface.Ridges or linear elevations can develop along the nail occurring in a“lengthwise” or “crosswise” direction. Beau's lines are lineardepressions that occur “crosswise” (transverse) in the fingernail.Leukonychia describes white streaks or spots on the nails. Kollonychiais an abnormal shape of the fingernail where the nail has raised ridgesand is thin and concave Kollonychia is often associated with irondeficiency.

Nail disorders which can be treated with the TNFα antibody of theinvention also include psoriatic nails. Psoriatic nails include changesin nails which are attributable to psoriasis. In some instancespsoriasis may occur only in the nails and nowhere else on the body.Psoriatic changes in nails range from mild to severe, generallyreflecting the extent of psoriatic involvement of the nail plate, nailmatrix, i.e., tissue from which the nail grows, nail bed. i.e., tissueunder the nail, and skin at the base of the nail. Damage to the nail bedby the pustular type of psoriasis can result in loss of the nail. Nailchanges in psoriasis fall into general categories that may occur singlyor all together. In one category of psoriatic nails, the nail plate isdeeply pitted, probably due to defects in nail growth caused bypsoriasis. In another category, the nail has a yellow to yellow-pinkdiscoloration, probably due to psoriatic involvement of the nail bed. Athird subtype of psoriatic nails are characterized by white areas whichappear under the nail plate. The white areas are actually air bubblesmarking spots where the nail plate is becoming detached from the nailbed. There may also be reddened skin around the nail. A fourth categoryis evidenced by the nail plate crumbling in yellowish patches, i.e.,onychodystrophy, probably due to psoriatic involvement in the nailmatrix. A fifth category is characterized by the loss of the nail in itsentirety due to psoriatic involvement of the nail matrix and nail bed.

Antibodies obtained using the method of the invention can also be usedto treat nail disorders often associated with lichen planus. Nails insubjects with lichen planus often show thinning and surface roughness ofthe nail plate with longitudinal ridges or pterygium.

The antibodies obtained using the invention can be used to treat naildisorders, such as those described herein. Often nail disorders areassociated with skin disorders. In one embodiment, the inventiontreatment for nail disorders using a TNFα antibody. In anotherembodiment, the nail disorder is associated with another disorder,including a skin disorder such as psoriasis. In another embodiment, thedisorder associated with a nail disorder is arthritis, includingpsoriatic arthritis.

12. Other Skin and Nail Disorders

Antibodies obtained using the method of the invention can be used totreat other skin and nail disorders, such as chronic actinic dermatitis,bullous pemphigoid, and alopecia areata. Chronic actinic dermatitis(CAD) is also referred to as photosensitivity dermatitis/actinicreticuloid syndrome (PD)/AR). CAD is a condition in which the skinbecomes inflamed, particularly in areas that have been exposed tosunlight or artificial light. Commonly, CAD patients have allergies tocertain substances that come into contact with their skin, particularlyvarious flowers, woods, perfumes, sunscreens and rubber compounds.Bullous pemphigoid refers to a skin disorder characterized by theformation of large blisters on the trunk and extremities. Alopeciaareata refers to hair loss characterized by round patches of completebaldness in the scalp or beard.

O. Vasculitides

TNFα has been implicated in the pathophysiology of a variety ofvasculitides, (see e.g., Deguchi et al. (1989) Lancet. 2:745). In oneembodiment, the invention provides a multiple-variable dose method forinhibiting TNFα activity in a subject suffering from a vasculitis inwhich TNFα activity is detrimental.

The term “vasculitis” or “vasculitides” as used interchangeably herein,refers to a group of disorders which are characterized by theinflammation of blood vessels. Blood vessels of all sizes may beaffected, from the largest vessel in the body (the aorta) to thesmallest blood vessels in the skin (capillaries). The size of bloodvessel affected varies according to the specific type of vasculitis. Asused her in, the term “a vasculitis in which TNFα activity isdetrimental” is intended to include vasculitis in which the presence ofTNFα in a subject suffering from the disorder has been shown to be or issuspected of being either responsible for the pathophysiology of thedisorder or a factor that contributes to a worsening of the disorder.Such disorders may be evidenced, for example, by an increase in theconcentration of TNFα in a biological fluid of a subject suffering fromthe disorder (e.g., an increase in the concentration of TNFα in serum,plasma, synovial fluid, etc. of the subject), which can be detected, forexample, using an anti-TNFα antibody as described above.

There are numerous examples of vasculitides in which TNFα activity isdetrimental, including Behect's disease. The use of the antibodies, orantigen-binding portions thereof, for treatment of specific vasculitidesis discussed further below. In certain embodiments, the antibody, orantibody portion, obtained using the invention is administered to thesubject in combination with another therapeutic agent, as describedbelow.

The antibody, or antibody portion, obtained using the invention may alsobe used to treat vasculitis in which TNFα activity is detrimental,wherein inhibition of TNFα activity is expected to alleviate thesymptoms and/or progression of the vasculitis or to prevent thevasculitis. Subjects suffering from or at risk of developing vasculitiscan be identified through clinical symptoms and tests. For example,subjects with vasculitides often develop antibodies to certain proteinsin the cytoplasm of neutrophils, antineutrophil cytoplasmic antibodies(ANCA). Thus, in some instances, vasculitides may be evidenced by tests(e.g., ELISA), which measure ANCA presence.

Vasculitis and its consequences may be the sole manifestation of diseaseor it may be a secondary component of another primary disease.Vasculitis may be confined to a single organ or it may simultaneouslyaffect several organs, and depending on the syndrome, arteries and veinsof all sizes can be affected. Vasculitis can affect any organ in thebody.

In vasculitis, the vessel lumen is usually compromised, which isassociated with ischemia of the tissues supplied by the involved vessel.The broad range of disorders that may result from this process is due tothe fact that any type, size and location of vessel (e.g., artery, vein,arteriole, venule, capillary) can be involved. Vasculitides aregenerally classified according to the size of the affected vessels, asdescribed below. It should be noted that some small and large vesselvasculitides may involve medium-sized arteries; but large andmedium-sized vessel vasculitides do not involve vessels smaller thanarteries. Large vessel disease includes, but is not limited to, giantcell arteritis, also known as temporal arteritis or cranial arteritis,polymyalgia rheumatica, and Takayasu's disease or arteritis, which isalso known as aortic arch syndrome, young female arteritis and Pulselessdisease. Medium vessel disease includes, but is not limited to, classicpolyarteritis nodosa and Kawasaki's disease, also known as mucocutaneouslymph node syndrome. Non-limiting examples of small vessel disease areBehcet's Syndrome, Wegner's granulomatosis, microscopic polyangitis,hypersensitivity vasculitis, also known as cutaneous vasculitis, smallvessel vasculitis, Henoch-Schonlein purpura, allergic granulamotosis andvasculitis, also known as Churg Strauss syndrome. Other vasculitidesinclude, but are not limited to, isolated central nervous systemvasculitis, and thromboangitis obliterans, also known as Buerger'sdisease. Classic Polyarteritis nodosa (PAN), microscopic PAN, andallergic granulomatosis are also often grouped together and are calledthe systemic necrotizing vasculitides. A further description ofvasculitis is described below:

1. Large Vessel Vasculitis

In one embodiment, the TNFα antibody obtained using the invention may beused to treat subjects who have large vessel vasculitis. The term “largevessel(s)” as used herein, refers to the aorta and the largest branchesdirected toward major body regions. Large vessels include, for example,the aorta, and its branches and corresponding veins, e.g., thesubclavian artery; the brachiocephalic artery; the common carotidartery; the innonimate vein; internal and external jugular veins; thepulmonary arteries and veins; the venae cavae; the renal arteries andveins; the femoral arteries and veins; and the carotid arteries.Examples of large vessel vasculitides are described below.

a. Giant Cell Arteritis (GCA)

Tumor necrosis factor has been implicated in the pathophysiology ofgiant cell arteritis (Sneller (2002) Cleve. Clin. J. Med. 69:SII40;Schett et al. (2002) Ann. Rheum. Dis. 61:463). Giant cell artentis(GCA), refers to a vasculitis involving inflammation and damage to bloodvessels, particularly the large or medium arteries that branch from theexternal carotid artery of the neck. GCA is also referred to as temporalarteritis or cranial arteritis, and is the most common primaryvasculitis in the elderly. It almost exclusively affects individualsover 50 years of age, however, there are well-documented cases ofpatients 40 years and younger. OCA usually affects extracranialarteries. OCA can affect the branches of the carotid arteries, includingthe temporal artery. GCA is also a systemic disease which can involvearteries in multiple locations.

Histopathologically, GCA is a panarteritis with inflammatory mononuclearcell infiltrates within the vessel wall with frequent Langhans typegiant cell formation. There is proliferation of the intima,granulomatous inflammation and fragmentation of the internal elasticlamina. The pathological findings in organs is the result of ischemiarelated to the involved vessels.

Patients suffering from GCA exhibit certain clinical symptoms, includingfever, headache, anemia and high erythrocyte sedimentation rate (ESR).Other typical indications of GCA include jaw or tongue claudication,scalp tenderness, constitutional symptoms, pale optic disc edema(particularly ‘chalky while’ disc edema), and vision disturbances. Thediagnosis is confirmed by temporal artery biopsy.

b. Polymyalgia Rheumatica

Tumor necrosis factor has been implicated in the pathophysiology ofpolymyalgia rheumatica (Straub et al. (2002) Rheumatology (Oxford)41:423; Uddhammar et al. (1998) Br. J. Rheumatol. 37:766). Polymyalgiarheumatica refers to a rheumatic disorder that is associated withmoderate to ever muscle pain and stiffness in the neck, shoulder, andhip, most noticeable in the morning. IL-6 and IL-1β expression has alsobeen detected in a majority of the circulating mloncmytes in patientswith the polymyalgia rheumatica. Polymyalgia rheumatica may occurindependently, or it may coexist with or precede GCA, which is aninflammation of blood vessels.

c. Takayasu's Arteritis

Tumor necrosis factor has been implicated in the pathophysiology ofTakayasu's arteritis (Kobayashi and Numano (2002) Intern. Med. 41:44;Fraga and Medina (2002) Curr. Rheumatol. Rep. 4:30). Takayasu'sarteritis refers to a vasculitis characterized by an inflammation of theaorta and its major branches. Takayasu's arteritis (also known as Aorticarch syndrome, young female arteritis and Pulseless disease) affects thethoracic and abdominal aorta and its main branches or the pulmonaryarteries. Fibrotic thickening of the aortic wall and its branches (e.g.,carotid, inominate, and subclavian arteries) can lead to reduction oflumen size of vessels that arise from the aortic arch. This conditionalso typically affects the renal arteries.

Takayasu's arteritis primarily affects young women, usually aged 20-40years old, particularly of Asian descent, and may be manifested bymalaise, arthralgias and the gradual onset of extremity claudication.Most patients have asymmetrically reduced pulses, usually along with ablood pressure differential in the arms. Coronary and/or renal arterystenosis may occur.

The clinical features of Takayasu's arteritis may be divided into thefeatures of the early inflammatory disease and the features of the laterdisease. The clinical features of the early inflammatory stage ofTakayasu's disease are: malaise, low grade fever, weight loss, myalgia,arthralgia, and erythema multiforme. Later stages of Takayasu's diseaseare characterized by fibrotic stenosis of arteries and thrombosis. Themain resulting clinical features are ischaemic phenomena, e.g. weak andasymmetrical arterial pulses, blood pressure discrepancy between thearms, visual disturbance, e.g. scotomata and hemianopia, otherneurological features including vertigo and syncope, hemiparesis orstroke. The clinical features result from ischaemia due to arterialstenosis and thrombosis.

2. Medium Vessel Disease

In one embodiment, the TNFα antibody obtained using the invention may beused to treat subjects who have medium vessel vasculitis. The term“medium vessel(s)” is used to refer to those blood vessels which are themain visceral arteries. Examples of medium vessels include themesenteric arteries and veins, the ilac arteries and veins, and themaxillary arteries and veins. Examples of medium vessel vasculitides aredescribed below.

a. Polyarteritis Nodosa

Tumor necrosis factor has been implicated in the pathophysiology ofpolyarteritis nodosa (DiGirolamo et al. (1997) J. Leukoc. Biol. 61:667).Polyarteritis nodosa, or periarteritis nodosa refers to vasculitis whichis a serious blood vessel disease in which small and medium-sizedarteries become swollen and damaged because they are attacked by rogueImmune cells. Polyarteritis nodosa usually affects adults morefrequently than children. It damages the tissues supplied by theaffected arteries because they don't receive enough oxygen andnourishment without a proper blood supply.

Symptoms which are exhibited in patients with polyarteritis nodosagenerally result from damage to affected organs, often the skin, heart,kidneys, and nervous system. Generalized symptoms of polyarteritisnodosa include fever, fatigue, weakness, loss of appetite, and weightloss. Muscle aches (myalgia) and joint aches (arthralgia, are common.The skin of subjects with polyarteritis nodosa may also show rashes,swelling, ulcers, and lumps (nodular lesions).

Classic PAN (polyarteritis nodosa) is a systemic arteritis of small tomedium muscular arteritis in which involvement of renal and visceralarteries is common. Abdominal vessels have aneurysms or occlusions in50% of PAN patients. Classic PAN does not involve the pulmonary arteriesalthough the bronchial vessels may be involved. Granulomas, significanteosinophilia and an allergic diathesis are not part of the syndrome.Although any organ system may be involved, the most commonmanifestations include peripheral neuropathy, mononeuritis multiplex,intestinal ischemia, renal ischemia, testicular pain and livedoreticutaris.

b. Kawasaki's Disease

Tumor necrosis factor has been implicated in the pathophysiology ofKawasaki's disease (Sundel (2002) Curr. Rheumatol. Rep. 4:474; Gedalia(2002) Curr. Rheumatol. Rep. 4:25). Although the cause of Kawasaki'sdisease is unknown, it is associated with acute inflammation of thecoronary arteries, suggesting that the tissue damage associated withthis disease may be mediated by proinflammatory agents such as TNFα.Kawasaki's disease refers to a vasculitis that affects the mucusmembranes, lymph nodes, lining of the blood vessels, and the heart.Kawasaki's disease is also often referred to as mucocutaneous lymph nodesyndrome, mucocutaneous lymph node disease, and infantile polyarteritis.Subjects afflicted with Kawasaki's disease develop vasculitis ofteninvolving the coronary arteries which can lead to myocarditis andpericarditis. Often as the acute inflammation diminishes, the coronaryarteries may develop aneurysm, thrombosis, and lead to myocardialinfarction.

Kawasaki's disease is a febrile systemic vasculitis associated withedema in the palms and the soles of the feet, with enlargement ofcervical lymph nodes, cracked lips and “strawberry tongue”. Although theinflammatory response is found in vessels throughout the body, the mostcommon site of end-organ damage is the coronary arteries. Kawasaki'sDisease predominantly affects children under the age of 5. The highestincidence is in Japan but is becoming increasingly recognized in theWest and is now the leading cause of acquired heart disease in USchildren. The most serious complication of Kawasaki disease is coronaryarteritis and aneurysm formation that occurs in a third of untreatedpatients.

3. Small Vessel Disease

In one embodiment, a TNFα antibody is used to treat subjects who havesmall vessel vasculitis. The term “small vessel(s)” is used to refer toarterioles, venules and capillaries. Arterioles are arteries thatcontain only 1 or 2 layers of sooth muscle cells and are terminal to andcontinuous with the capillary network. Venules carry blood from thecapillary network to veins and capillaries connect arterioles andvenules.

Examples of small vessel vasculitides are described below.

a. Behcet's Disease

Tumor necrosis factor has been implicated in the pathophysiology ofBehcet's disease (Sfikakis (2002) Ann. Rheum. Dis. 61:1151-3; Dogan andFarah (2002) Oftalmologia. 52:23). Behcet's disease is a chronicdisorder that involves inflammation of blood vessels throughout thebody. Behcet's disease may also cause various types of skin lesions,arthritis, bowel inflammation, and meningitis (inflammation of themembranes of the brain and spinal cord). As a result of Behcet'sdisease, the subject with the disorder may have inflammation in tissuesand organs throughout the body, including the gastrointestinal tract,central nervous system, vascular system, lungs, and kidneys. Behcet'sdisease is three times more common in males than females and is morecommon in the eastern Mediterranean and Japan.

Subjects who have Behcet's disease may show clinical symptoms includingrecurrent oral ulcers (resembling canker sores), recurrent genitalulcers, and eye inflammation. Serum levels of TNFα, IL-8, IL-1, IL-6INT-γ and IL-12 are elevated in Behcet's patients, and the production ofthese factors has been shown to be elevated in the monocytes of Behcet'spatients (see. e.g., Inflammatory Disease of Blood Vessels (2001) MarcelDekker, Inc., eds. G. S. Hoffman and C. M. Weyand, p. 473).

b. Wegener's Granulomatosis

Tumor necrosis factor has been implicated in the pathophysiology ofWegener's granulomatosis (Marquez et al. (2003) Curr. Rheumatol. Rep.5:128; Harman and Margo (1998) Surv. Ophthalmol. 42:458). Wegener'sgranulomatosis refers to a vasculitis that causes inflammation of bloodvessels in the upper respiratory tract (nose, sinuses, ears), lungs, andkidneys. Wegener's granulomatosis is also referred to as midlinegranulomatosis. Wegener's granulomatosis includes a granulomatousinflammation involving the respiratory tract, and necrotizing vasculitisaffecting small to medium-sized vessels. Subjects who have Wegener'sgranulomatosis often also have arthritis (joint inflammation).Glomerulonephritis may also be present in affected subjects, butvirtually any organ may be involved.

Patients affected with Wegener's granulomatosis typically show clinicalsymptoms comprising recurrent sinusitis or epistaxis, mucosalulcerations, otitis media, cough, hemoptysis and dyspnea. The firstsymptoms of Wegener's granulomatosis frequently include upperrespiratory tract symptoms, joint pains, weakness, and tiredness.

c. Churg-Strauss syndrome

Tumor necrosis factor has been implicated in the pathophysiology ofChurg-Strauss syndrome (Gross (2002) Curr. Opin. Rheumatol. 14:11; Churg(2001) Mod. Pathol. 14:1284). Churg-Strauss syndrome refers to avasculitis that is systemic and shows early manifestation signs ofasthma and eosinophilia. Churg-Strauss syndrome is also referred to asallergic granulomatosis and angitis, and occurs in the setting ofallergic rhinitis, asthma and eosinophilia. Sinusitis and pulmonaryinfiltrates also occur in Churg-Strauss syndrome, primarily affectingthe lung and heart. Peripheral neuropathy, coronary arteritis andgastrointestinal involvement are common.

Patients afflicted with Churg-Strauss syndrome can be diagnosedaccording to criteria established by the American College ofRheumatology (ACR). These criteria were intended to distinguish CSS fromother forms of vasculitis. Not all patients meet every criterion. Some,in fact, may have only 2 or 3 criteria, yet they are still classified asChurg-Strauss syndrome. The ACR selected 6 disease features (criteria)as being those that best distinguished Churg-Strauss syndrome frontother vasculitides. These criteria include: 1) asthma; 2) eosinophilia[>10% on differential WBC count]; 3) mononeuropathy; 4) transientpulmonary infiltrates on chest X-rays: 5) paranasal sinus abnormalities;and 6) biopsy comprising a blood vessel with extravascular eosinophils.

P. Other TNFα-Related Disorders

In one embodiment, the invention features a multiple-variable dosemethod for treating a TNFα-related disorder in which TNFα activity isdetrimental, comprising administering to a subject a TNFα antibody, suchthat said TNFα-related disorder is treated. Examples of TNFα-relateddisorders in which TNFα activity is detrimental, are discussed furtherbelow.

1. Juvenile Arthritis

Tumor necrosis factor has been implicated in the pathophysiology ofjuvenile arthritis, including juvenile rheumatoid arthritis (Grom et al.(1996) Arthritis Rheum. 39:1703; Mangge et al. (1995) Arthritis Rheum.8:211). In one embodiment, the TNFα antibody of the invention is used totreat juvenile rheumatoid arthritis.

The term “juvenile rheumatoid arthritis” or “JRA” as used herein refersto a chronic, inflammatory disease which occurs before age 16 that maycause joint or connective tissue damage. JRA is also referred to asjuvenile chronic polyarthritis and Stills disease.

JRA causes joint inflammation and stiffness for more than 6 weeks in achild of 16 years of age or less. Inflammation causes redness, swelling,warmth, and soreness in the joints. Any joint can be affected andinflammation may limit the mobility of affected joints. One type of JRAcan also affect the internal organs.

JRA is often classified into three types by the number of jointsinvolved, the symptoms, and the presence or absence of certainantibodies found by a blood test. These classifications help thephysician determine how the disease will progress and whether theinternal organs or skin is affected. The classifications of JRA includethe following

a. Pauciarticular JRA, wherein the patient has four or fewer joints areaffected. Pauciarticular is the most common form of JRA, and typicallyaffects large joints, such as the knees.

b. Polyarticular JRA, wherein live or more joints are affected. Thesmall joints, such as those in the hands and feet, are most commonlyinvolved, but the disease may also affect large joints.

c. Systemic JRA is characterized by joint swelling, fever, a light skinrash, and may also affect internal organs such as the heart, liver,spleen, and lymph nodes. Systemic JRA is also referred to as it Still'sdisease. A small percentage of these children develop arthritis in manyjoints and can have severe arthritis that continues into adulthood.

2. Endometriosis

Tumor necrosis factor has been implicated in the pathophysiology ofendometriosis, as women with endometriosis have elevated peritoneallevels of TNF (Eisermann et al. (1988) Fertil Steril 50:573; Halme(1989) Am J Obstet Gynecol 161:1718; Mori et al. (1991) Am J ReprodImmunol 26:62: Taketani et al. (1992) Am J Obstet Gynecol 167:265;Overton et al. (1996) Hum Reprod 1996; 11:380). In one embodiment, theTNFα antibody may be used to treat endometriosis. The term“endometriosis” as used herein refers to a condition in which the tissuethat normally lines the uterus (endometrium) grows in other areas of thebody, causing pain, irregular bleeding, and frequently infertility.

3. Prostatitis

Tumor necrosis factor has been implicated in the pathophysiology ofprostatitis, as men with chronic prostatitis and chronic pelvic painhave significantly higher levels of TNF and IL-1 in semen compared tocontrols (Alexander et al. (1998) Urology 52:744; Nadler et al. (2000) JUrol 164:214; Orhan et al. (2001) Int J Urol 8:495) Furthermore, in arat model of prostatitis TNF levels were also increased in comparison tocontrols (Asakawa et al. (2001) Hinyokika Kiyo 47:459; Harris et al.(2000) Prostate 44:25). In one embodiment, the TNFα antibody of theinvention is used to treat prostatitis.

The term “prostatitis” as used herein refers to an inflammation of theprostate. Prostatitis is also referred to as pelvic pain syndrome.Prostatitis manifests itself in a variety of forms, includingnonbacterial prostatitis, acute prostatitis, bacterial prostatitis, andacute prostatitis. Acute prostatitis refers to an inflammation of theprostate gland that develops suddenly. Acute prostatitis is usuallycaused by a bacterial infection of the prostate gland. Chronicprostatitis is an inflammation of the prostate gland that developsgradually, continues for a prolonged period, and typically has subtlesymptoms. (Chronic prostatitis is also usually caused by a bacterialinfection

4. Choroidal Neovascularization

Tumor necrosis factor has been implicated in the pathophysiology ofchoroidal neovascularization. For example, in surgically excisedchoroidal neovascular membranes, neovascular vessels stained positivefor both TNF and IL-1 (Oh H et al. (1999) Invest Ophthalmol Vis Sci40:1891). In one embodiment, the TNFα antibody is used to treatchoroidal neovascularization. The term “choroidal neovascularization” asused herein refers to the growth of new blood vessels that originatefrom the choroid through a break in the Bruch membrane into thesub-retinal pigment epithelium (sub-RPE) or subretinal space. Choroidalneovascularization (CNV) is a major cause of visual loss in patientswith the condition.

5. Sciatica

Tumor necrosis factor has been implicated in the pathophysiology ofsciatica (Ozaktay et al. (2002) Eur Spine J. 11:467; Brisby et al.(2002) Eur Spine J. 11:62). In one embodiment, the TNFα antibody of theinvention is used to treat sciatica. The term “sciatica” as used hereinrefers to a condition involving impaired movement and/or sensation inthe leg, caused by damage to the sciatic nerve. Sciatica is alsocommonly referred to as neuropathy of the sciatic nerve and sciaticnerve dysfunction. Sciatica is a form of peripheral neuropathy. Itoccurs when there is damage to the sciatic nerve, located in the back ofthe leg. The sciatic nerve controls the muscles of the back of the kneeand lower leg and provides sensation to the back of the thigh, part ofthe lower leg and the sole of the foot. Sciatica can be indicative ofanother disorder, including a lumbar herniated disc, spinal stenosis,degenerative disc disease, isthmic spondyloisthesis and piniformissyndrome.

6. Sjogren's Syndrome

Tumor necrosis factor has been implicated in the pathophysiology ofSjogren's syndrome (Koski et al. (2001) Clin Exp Rheumatol. 19:131). Inone embodiment, the TNFα antibody of the invention is used to treatSjogren's syndrome. The term “Sjogren's syndrome” as used herein refersto a systemic inflammatory disorder characterized by dry mouth,decreased tearing, and other dry mucous membranes, and is oftenassociated with autoimmune rheumatic disorders, such as rheumatoidarthritis. Dryness of the eyes and mouth are the most common symptoms ofthis syndrome. The symptoms may occur alone, or with symptoms associatedwith rheumatoid arthritis or other connective tissue diseases. There maybe an associated enlargement of the salivary glands. Other organs maybecome affected. The syndrome may be associated with rheumatoidarthritis, systemic lupus erythematosus, scleroderma, polymyositis, andother diseases.

7. Uveitis

Tumor necrosis factor has been implicated in the pathophysiology ofuveitis (Wakefield and Lloyd (1992) Cytokine 4:1; Woon et al. (1998)Curr Eye Res. 17:955). In one embodiment, the TNFα antibody of theinvention is used to treat uveitis. The term “uveitis” as used hereinrefers to an inflammation of the uvea, which is the layer between thesclera and the retina, which includes the iris, ciliary body, and thechoroid. Uveitis is also commonly referred to as iritis, pars planitis,chroiditis, chorioretinitis, anterior uveitis, and posterior uveitis.The most common form of uveitis is anterior uveitis, which involvesinflammation in the front part of the eye, which is usually isolated tothe iris. This condition is often called iritis. In one embodiment, theterm uveitis refers to an inflammation of the uvea which excludesinflammation associated with an autoimmune disease, i.e., excludesautoimmune uveitis.

8. Wet Macular Degeneration

Tumor necrosis factor has been implicated in the pathophysiology of wetmacular degeneration. In one embodiment, the TNFα antibody of theinvention is used to treat wet macular degeneration. The term “wetmacular degeneration” as used herein refers to a disorder that affectsthe macula (the central part of the retina of the eye) and causesdecreased visual acuity and possible loss of central vision. Patientswith wet macular degeneration develop new blood vessels under theretina, which causes hemorrhage, swelling, and scar tissue.

9. Osteoporosis

Tumor necrosis factor has been implicated in the pathophysiology ofosteoporosis, (Tsutsumimoto et al. (1999) J Bone Miner Res. 14:1751).Osteoporosis is used to refer to a disorder characterized by theprogressive loss of bone density and thinning of bone tissue.Osteoporosis occurs when the body fails to form enough new bone, or whentoo much old bone is reabsorbed by the body, or both. The TNFα antibody,or antigen-binding fragment thereof, of the invention can be used totreat osteoporosis.

10. Osteoarthritis

Tumor necrosis factor has been implicated in the pathophysiology ofosteoarthritis, (Venn et al. (1993) Arthritis Rheum. 36:819, Westacottet al. (1994) J Rheumatol. 21:1710). Osteoarthritis (OA) is alsoreferred to as hypertrophic osteoarthritis, osteoarthrosis, anddegenerative joint disease. OA is a chronic degenerative disease ofskeletal joints, which affects specific joints, commonly knees, hips,hand joints and spine, in adults of all ages. OA is characterized by anumber of the following manifestations including degeneration andthinning of the articular cartilage with associated development of“ulcers” or craters, osteophyte formation, hypertrophy of bone at themargins, and changes in the snyovial membrane and enlargement ofaffected joints. Furthermore, osteoarthritis is accompanied by pain andstiffness, particularly after prolonged activity. The antibody, orantigen-binding fragment thereof, of the invention can be used to treatosteoarthritis. Characteristic radiographic features of osteoarthritisinclude joint space narrowing, subchondral sclerosis, osteophytosis,subchondral cyst formation, loose osseous body (or “joint mouse”).

Medications used to treat osteoarthritis include a variety ofnonsteroidal, anti-inflammatory drugs (NSAIDs). In addition, COX 2inhibitors, including Celebrex. Vioxx, and Bextra, and Etoricoxib, arealso used to treat OA. Steroids, which are injected directly into thejoint, may also be used to reduce inflammation and pain. In oneembodiment of the invention, TNFα antibodies of the invention areadministered in combination with a NSAIDs, a COX2 inhibitor, and/orsteroids.

11. Other

The methods of the invention, also can be used to treat various otherdisorders in which TNFα activity is detrimental. Examples of otherdiseases and disorders in which TNFα activity has been implicated in thepathophysiology, and thus which can be treated using an antibody, orantibody portion, of the invention, include inflammatory bone disorders,bone resorption disease, coagulation disturbances, burns, reperfusioninjury, keloid formation, scar tissue formation, pyrexia, periodontaldisease, obesity, radiation toxicity, age-related cachexia, Alzheimer'sdisease, brain edema, inflammatory brain injury, cancer, chronic fatiguesyndrome, dermatomyositis, drug reactions, such as Stevens-Johnsonsyndrome and Jarisch-Herxheimer reaction, edema in and/or around thespinal cord, familial periodic fevers, Felty's syndrome, fibrosis,glomerulonephritides (e.g. post-streptococcal glomerulonephritis or IgAnephropathy), loosening of prostheses, microscopic polyangitis, mixedconnective tissue disorder, multiple myeloma, cancer and cachexia,multiple organ disorder, myelo dysplastic syndrome, orchitismosteolysis, pancreatitis, including acute, chronic, and pancreaticabscess, polymyositis, progressive renal failure, pseudogout, pyodermagangrenosum, relapsing polychondritis, rheumatic heart disease,sarcoidosis, sclerosing cholangitis, stroke, thoracoabdominal aorticaneurysm repair (TAAA), TNF receptor associated periodic syndrome(TRAPS), symptoms related to Yellow Fever vaccination, inflammatorydiseases associated with the ear, chronic ear inflammation, chronicotitis media with or without cholesteatoma, pediatric ear inflammation,myotosis, ovarian cancer, colorectal cancer, therapy associated withinduced inflammatory syndrome (e.g., syndromes following IL-2administration), and a disorder associated with a reperfussion injury.

It is understood that all of the above-mentioned TNFα-related disordersinclude both the adult and juvenile forms of the disease whereappropriate. It is also understood that all of the above-mentioneddisorders include both chronic and acute forms of the disease. Inaddition, the multiple-variable dose methods of the invention can beused to treat each of the above-mentioned TNFα-related disorders aloneor in combination with one another, e.g., a subject who is sufferingfrom uveitis and lupus.

Additional Therapeutic Agents

The invention pertains to pharmaceutical compositions and methods of usethereof for the treatment of a TNFα-related disorder using amultiple-variable dose regimen. The pharmaceutical compositions comprisea first agent that prevents or inhibits a TNFα-related disorder. Thepharmaceutical composition and methods of use may comprise a secondagent that is an active pharmaceutical ingredient; that is, the secondagent is therapeutic and its function is beyond that of an inactiveingredient, such as a pharmaceutical carrier, preservative, diluent, orbuffer. The second agent may be useful in treating or preventingTNFα-related disorders. The second agent may diminish or treat at leastone symptom(s) associated with the targeted disease. The first andsecond agents may exert their biological effects by similar or unrelatedmechanisms of action; or either one or both of the first and secondagents may exert their biological effects by a multiplicity ofmechanisms of action. A pharmaceutical composition may also comprise athird compound, or even more yet, wherein the third (and fourth, etc.)compound has the same characteristics of a second agent.

It should be understood that the pharmaceutical compositions describedherein may have the first and second, third, or additional agents in thesame pharmaceutically acceptable carrier or in a differentpharmaceutically acceptable carrier for each described embodiment. Itfurther should be understood that the first, second, third andadditional agent may be administered simultaneously or sequentiallywithin described embodiments. Alternatively, a first and second agentmay be administered simultaneously, and a third or additional agent maybe administered before or after the first two agents.

The combination of agents used within the methods and pharmaceuticalcompositions described herein may have a therapeutic additive orsynergistic effect on the condition(s) or disease(s) targeted fortreatment. The combination of agents used within the methods orpharmaceutical compositions described herein also may reduce adetrimental effect associated with at least one of the agents whenadministered alone or without the other agent(s) of the particularpharmaceutical composition. For example, the toxicity of side effects ofone agent may be attenuated by another agent of the composition, thusallowing a higher dosage, improving patient compliance, and improvingtherapeutic outcome. The additive or synergistic effects, benefits, andadvantages of the compositions apply to classes of therapeutic agents,either structural or functional classes, or to individual compoundsthemselves.

Supplementary active compounds can also be incorporated into thecompositions. In certain embodiments, an antibody or antibody portion ofthe invention is coformulated with and/or coadministered with one ormore additional therapeutic agents that are useful for treatingTNFα-related disorder in which TNFα activity is detrimental. Forexample, an anti-hTNFα antibody, antibody portion, or other TNFαinhibitor of the invention may be coformulated and/or coadministeredwith one or more additional antibodies that bind other targets (e.g.,antibodies that bind other cytokines or that bind cell surfacemolecules), one or more cytokines, soluble TNFα receptor (see e.g., PCTPublication No. WO 94/06476) and/or one or more chemical agents thatinhibit hTNFα production or activity (such as cyclohexane-ylidenederivatives as described in PCT Publication No. WO 93/19751).Furthermore, one or more antibodies or other TNFα inhibitors of theinvention may be used in combination with two or more of the foregoingtherapeutic agents. Such combination therapies may advantageouslyutilize lower dosages of the administered therapeutic agents, thusavoiding possible toxicities or complications associated with thevarious monotherapies. Specific therapeutic agent(s) are generallyselected based on the particular TNFα-related disorder being treated, asdiscussed below.

Nonlimiting examples of therapeutic agents with which an antibody,antibody portion, or other TNFα inhibitor can be combined in a multiplevariable dose method of treatment of the invention include thefollowing: non-steroidal anti-inflammatory drug(s) (NSAIDs); cytokinesuppressive anti-inflammatory drug(s) (CSAIDs); CDP-571/BAY-10-3356(humanized anti-TNFα antibody; Celltech/Bayer); cA2/infliximab (chimericanti-TNFα antibody; Centocor); 75 kdTNFR-IgG/etanercept (75 kD TNFreceptor-IgG fusion protein; Immunex; see e.g. Arthritis & Rheumatism(1994) Vol. 37, S295; J. Invest. Med. (1996) Vol. 44, 235A); 55kdTNF-IgG (55 kD TNF receptor-IgG fusion protein; Hoffmann-LaRoche)IDEC-CE9.1/SB 210396 (non-depleting primatized anti-CD4 antibody;IDEC/SmithKline; see e.g., Arthritis & Rheumatism (1995) Vol. 38, S185);DAB 486-IL-2 and/or DAB 389-IL-2 (IL-2 fusion proteins; Seragen; seee.g., Arthritis & Rheumatism (1993) Vol. 36, 1223); Anti-Tac (humanizedanti-IL-2Rα; Protein Design Labs/Roche); IL-4 (anti-inflammatorycytokine; DNAX/Schering); IL-10 (SCH 52000; recombinant IL-10,anti-inflammatory cytokine; DNAX/Schering); IL-4; IL-10 and/or IL-4agonists (e.g., agonist antibodies); IL-1RA (IL-1 receptor antagonist;Synergen/Angen); anakinra (Kineret®/Amgen); TNF-bp/s-TNF (soluble TNFbinding protein; see e.g., Arthritis & Rheumatism (1996) Vol. 39, No. 9(supplement), S284; Amer. J. Physiol. —Heart and Circulatory Physiology(1995) Vol. 268, pp. 37-42); R973401 (phosphodiesterase Type IVinhibitor; see e.g., Arthritis & Rheumatism (1996) Vol. 39. No. 9(supplement), S282); MK-966 (COX-2 Inhibitor; see e.g., Arthritis &Rheumatism (1996) Vol. 3.9, No. 9 (supplement), S81); Iloprost (seee.g., Arthritis & Rheumatism (1996) Vol. 39, No. 9 (supplement), S82);methotrexate; thalidomide (see e.g., Arthritis & Rheumatism (1996) Vol.39, No. 9 (supplement), S282) and thalidomide-related drugs (e.g.,Celgen); leflunomide (anti-inflammatory and cytokine inhibitor; seee.g., Arthritis & Rheumatism (1996) Vol. 39, No. 9 (supplement), S131;Inflammation Research (1996) Vol. 45, pp. 103-107); tranexamic acid(inhibitor of plasminogen activation; see e.g., Arthritis & Rheumatism(1996) Vol. 39. No. 9 (supplement), S284); T-614 (cytokine inhibitor;see e.g., Arthritis & Rheumatism (1996) Vol. 39, No. 9 (supplement),S282); prostaglandin E1 (see e.g., Arthritis & Rheumatism (1996) Vol.39, No. 9 (supplement), S282); Tenidap (non-steroidal anti-inflammatorydrug; see e.g., Arthritis & Rheumatism (1996) Vol. 39. No. 9(supplement), S280); Naproxen (non-steroidal anti-inflammatory drug; seee.g., Neuro Report (1996) Vol. 7, pp. 1209-1213); Meloxicam(non-steroidal anti-inflammatory drug); Ibuprofen (non-steroidalanti-inflammatory drug); Piroxicam (non-steroidal anti-inflammatorydrug); Diclofenac (non-steroidal anti-inflammatory drug); Indomethacin(non-steroidal anti-inflammatory drug); Sulfasalazine (see e.g.,Arthritis & Rheumatism (1996) Vol. 39. No. 9 (supplement), S281);Azathioprine (see e.g., Arthritis & Rheumatism (1996) Vol. 39, No. 9(supplement), S281); ICE inhibitor (inhibitor of the enzymeinterleukin-1β converting enzyme); zap-70 and/or lck inhibitor(inhibitor of the tyrosine kinase zap-70 or lck); VEGF inhibitor and/orVEGF-R inhibitor (inhibitors of vascular endothelial cell growth factoror vascular endothelial cell growth factor receptor; inhibitors ofangiogenesis); corticosteroid anti-inflammatory drugs (e.g., SB203580);TNF-convertase inhibitors; anti-IL-12 antibodies; ant-IL-18 antibodies;interleukin-11 (see e.g., Arthritis & Rheumatism (1996) Vol. 39, No. 9(supplement), S296); interleukin-13 (see e.g., Arthritis & Rheumatism(1996) Vol. 39, No. 9 (supplement), S308); interleukin-17 inhibitors(see e.g., Arthritis & Rheumatism (1996) Vol. 39, No. 9 (supplement),S120); gold; penicillamine; chloroquine; hydroxychloroquine;chlorambucil; cyclosporine; cyclophosphamide; total lymphoidirradiation; anti-thymocyte globulin; anti-CD4 antibodies; CD5-toxins;orally administered peptides and collagen; lobenzarit disodium; CytokineRegulating Agents (CRAs) HP228 and HP466 (Houghten Pharmaceuticals,Inc.); ICAM-1 antisense phosphorothioate oligodeoxynucleotides (ISIS2302; Isis Pharmaceuticals, Inc.); soluble complement receptor 1 (TP10;T Cell Sciences. Inc.); prednisone; orgotein; glycosaminoglycanpolysulphate; minocycline; anti-IL2R antibodies; marine and botanicallipids (fish and plant seed fatty acids; see e.g., DeLuca et al. (1995)Rheum. Dir. Clin. North Am. 21:759-777); auranofin; phenylbutazone;meclofenamic acid; flufenamic acid; intravenous immune globulin;zileuton; azaribine; mycophenolic acid (RS-61443); tacrolimus (FK-506);sirolimus (rapamycin); amiprilose (therafectin); cladribine(2-chlorodeoxyadenosine); methotrexate; antivirals; and immunemodulating agents. Any of the above-mentioned agents can be administeredin combination with the TNFα antibody of the invention to treat anTNFα-related disorder using the multiple variable dose or single dosemethod of treatment; of the invention.

In one embodiment, the TNFα antibody of the invention is administered incombination with one of the following agents for the treatment ofrheumatoid arthritis using the multiple variable dose method oftreatment of the invention: small molecule inhibitor of KDR (ABT-123),small molecule inhibitor of Tie-2; methotrexate; prednisone; celecoxib;folic acid; hydroxychloroquine sulfate; rofecoxib; etanercept;infliximab; anakinra (Kineret®/Amgen); leflunomide; naproxen;valdecoxib; sulfasaiazine; ibuprofen; methylprednisolone meloxicam;methylprednisolone acetate; gold sodium thiomalate; aspirin;azathioprine; triamcinolone acetonide; propxyphene napsylate/apap;folate; nabumetone; diclofenac; piroxicam; etodolac; diclofenac sodium;oxaprozin; oxycodone hcl; hydrocodone bitartrate/apap; diclofenacsodium/misoprostol; fentanyl; anakinra, human recombinant; tramadol hcl;salsalate; sulindac; cyanocobalamin/fa/pyridoxine; acetaminophen;alendronate sodium; prednisolone; morphine sulfate; lidocainehydrochloride; indomethacin; glucosamine sulfate/chondroitin;cyclosporine; sulfadiazine; amitriptyline hel; oxycodonehel/acetaminophen; olopatadine hel; misoprostol; naproxen sodium;omeprazole; mycophenolate mofetil; cyclophosphamide; rituximab; IL-1TRAP; MRA; CTLA4-IG; IL-18 BP; ABT-874; ABT-325 (anti-IL 18); anti-IL15; BIRB-796; SCTO-469; VX-702; AMG-548; VX-740; Roflumilast; IC-485;CDC-801; and mesopram. In another embodiment, the TNFα antibody of theinvention is administered using a multiple-variable dose method for thetreatment of a TNFα related disorder in combination with one of theabove mentioned agents for the treatment of rheumatoid arthritis. Inanother embodiment, the above-mentioned additional agents are used incombination with a TNFα antibody in the single dose method of treatmentof the invention.

In one embodiment, the TNFα antibody of the invention is administeredusing the multiple variable dose regimen in combination with one of thefollowing agents for the treatment of a TNFα-related disorder in whichTNFα activity is detrimental; anti-IL12 antibody (ABT 874); anti-IL18antibody (ABT 325); small molecule inhibitor of LCK; small moleculeinhibitor of COT; anti-IL1 antibody; small molecule inhibitor of MK2;anti-CD19 antibody; small molecule inhibitor of CXCR3; small moleculeinhibitor of CCR5; small molecule inhibitor of CCR11 anti-E/L selectinantibody; small molecule inhibitor of P2X7; small molecule inhibitor ofIRAK-4; small molecule agonist of glucocorticoid receptor; anti-C5areceptor antibody; small molecule inhibitor of C5a receptor; anti-CD32antibody; and CD32 as a therapeutic protein.

In yet another embodiment, a TNFα antibody obtained using the inventionmay be administered in combination with an antibiotic or antiinfectiveagent. Antiinfective agents include those agents known in the art totreat viral, fingal, parasitic or bacterial infections. The term,“antibiotic,” as used herein, refers to a chemical substance thatinhibits the growth of, or kills, microorganisms. Encompassed by thisterm are antibiotic produced by a microorganism, as well as syntheticantibiotics (e.g., analogs) known in the art. Antibiotics include, butare not limited to, clarithromycin (Biaxin®), ciprofloxacin (Cipro®),and metronidazole (Flagyl®).

In another embodiment, a TNFα antibody obtained using the invention maybe administered with an additional therapeutic agent to treat sciaticaor pain. Examples of agents which can be used to reduce or inhibit thesymptoms of sciatica or pain include hydrocodone bitartrate/apap,rofecoxib, cyclobenzaprine hcl, methylprednisolone, naproxen, ibuprofen,oxycodone hcl/acetaminophen, celecoxib, valdecoxib, methylprednisoloneacetate, prednisone, codeine phosphate/apap, tramadol hcl/acetaminophen,metaxalone, meloxicam, methocarbamol, lidocaine hydrochloride,diclofenac sodium, gahapentin, dexamethasone, carisoprodol, ketorolactromethamine, indomethacin, acetaminophen, diazepam, nabumetone,oxycodone hel, tizanidine hcl, diclofenac sodium/misoprostol,propoxyphene napsylate/apap, asa/oxycod/oxycodone ter,ibuprofen/hydrocodone bit, tramadol hcl, etodolac, propoxyphene hcl,amitriptyline hcl, carisoprodol/codeine phos/asa, morphine sulfate,multivitamins, naproxen sodium, orphenadrine citrate, and temazepam.

In yet another embodiment, the TNFα-related disorder is treated with aTNFα antibody obtained using the invention in combination withhemodialysis.

In another embodiment, a TNFα antibody obtained using the invention maybe used in combination with a drug used to treat Crohn's disease or aCrohn's-related disorder in the multiple variable dose regimen of theinvention. Examples of therapeutic agents which can be used to treatCrohn's include mesalamine, prednisone, azathioprine, mercaptopurine,infliximab, budesonide, sulfasalazine, methylprednisolone sod succ,diphenoxylate/atrop sulf, loperamide hydrochloride, methotrexate,omeprazole, folate, ciprofloxacin/dextrose-water, hydrocodonebitartrate/apap, tetracycline hydrochloride, fluocinonide,metronidazole, thimerosal/boric acid, hyoscyamine sulfate,cholestyramine/sucrose, ciprofloxacin hydrochloride, meperidinehydrochloride, midazolam hydrochloride, oxycodone hcl/acetaminophen,promethazine hydrochloride, sodium phosphate,sulfamethoxazole/trimethoprim, celecoxib, polycarbophil, propoxyphenenapsylate, hydrocortisone, multivitamins, balsalazide disodium, codeinephosphate/apap, colesevelam hel, cyanocobalamin, folic acid,levofloxacin, matalizumab, methylprednisolone, interferon-gamma, andsargramostim (GM-CSF). In one embodiment, methotrexate is administeredfor the treatment of Crohn's disease at a dose of 2.5 mg to 30 mg perweek.

In another embodiment, a TNFα antibody is administered in combinationwith an additional therapeutic agent to treat asthma in the multiplevariable dose regimen of the invention. Examples of agents which can beused to reduce or inhibit the symptoms of asthma include the following:albuterol; salmeterol/fluticasone; sodium; fluticasone propionate;budesonide; prednisone; salmeterol xinafoate; levalbuterol hcl;sulfate/ipratropium; prednisolone sodium phosphate; triamcinoloneacetonide; beclomethasone dipropionate; ipratropium bromide;Azithromycin; pirbuterol acetate; prednisolone; theophylline anhydrous;zafirlukast; methylprednisolone sod succ; clarithromycin; formoterolfumarate; influenza virus vaccine; methylprednisolone; trihydrate;allergy injection cromolyn sodium; cefprozil; fexofenadinehydrochloride; flunisolide/menthol; levofloxacin;amoxicillin/clavulanate, inhaler assist device, guaifenesin,dexamethasone sod phosphate; moxifloxacin hel; hyclate;guaifenesin/d-methorphan; gatifloxacin; pephedrine/cod/chlorophenir;cetirizine hydrochloride; mometasone furoate; salmeterol xinafoate;benzonatate; cephalexin; pe/hydrocodone/chlorphenir, cetirizinehcl/pseudoephed; phenylephrine/cod/promethazine; codeine/promethazine;flunisolide; dexamethasone; guaifenesin/pseudoephedrine;chlorpheniramine/hydrocodone; nedoeromil sodium; terbutaline sulfate;epinephrine and methylprednisolone, metaproterenol sulfate.

In another embodiment, the TNFα □ antibody of the invention isadministered in combination with an additional therapeutic agent totreat COPD. Examples of agents which can be used to reduce or inhibitthe symptoms of COPD include, albuterol sulfate/ipratropium; ipratropiumbromide; salmeterol/fluticasone; albuterol; salmeterol; xinafoate;fluticasone propionate; prednisone; theophylline anhydrous;levofloxacin; methylprednisolone sod succ; montelukast sodium;budesonide; formoterol fumarate; triamcinolone acetonide; guaifenesin;azithromycin; beclomethasone; dipropionate; levalbuterol hel;flunisolide; sodium; trihydrate; gatifloxacin; zafirlukast; furoate;amoxicillin/clavulanate; flunisolide/menthol;chlorpheniramine/hydrocodone; metaproterenol sulfate;methylprednisolone; ephedrine/cod/chlorphenir, pirbuterol acetate;-ephedrine/loratadine; terbutaline sulfate; tiotropium bromide;(R,R)-formoterol; TgAAT; Cilomilast and Roflumilast

In another embodiment, the TNFα antibody of the invention isadministered in combination with an additional therapeutic agent totreat IPF. Examples of agents which can be used to reduce or inhibit thesymptoms of IPF include prednisone; azathioprine; albuterol;colchicines; sulfate; digoxin; gamma interferon; methylprednisolone sodsucc; furosemide; lisinopril; nitroglycerin; spironolactone;cyclophosphamide; ipratropium bromide; actinomycin d; alteplase;fluticasone propionate; levofloxacin; metaproterenol sulfate; morphinesulfate; oxycodone hcl; potassium chloride; triamcinolone acetonide;tacrolimus anhydrous; calcium; interferon-alpha; methotrexate;mycophenolate mofetil.

In one embodiment of the invention, a TNFα antibody is administered incombination with an agent which is commonly used to treatspondyloarthropathies. Examples of such agents include nonsteroidal,anti-inflammatory drugs (NSAIDs), COX inhibitors, including Celebrex®,Vioxx®, and Bextra®, and etoricoxib. Physiotherapy is also commonly usedto treat spondyloarthropathies, usually in conjunction with non-steoidalinflammatory drugs.

In another embodiment, the TNFα antibody of the invention may beadministered in combination with an additional therapeutic agent totreat ankylosing spondylitis. Examples of agents which can be used toreduce or inhibit the symptoms of ankylosing spondylitis includeibuprofen, diclofenac and misoprostol, naproxen, meloxicam,indomethacin, diclofenac, celecoxib, rofecoxib, sulfasalazine,prednisone, methotrexate, azathioprine, minocyclin, prednisone,etanercept, and infliximab.

In another embodiment, the TNFα antibody of the invention isadministered in combination with an additional therapeutic agent totreat psoriatic arthritis. Examples of agents which can be used toreduce or inhibit the symptoms of psoriatic arthritis includemethotrexate; etanercept; rofecoxib; celecoxib; folic acid;sulfasalazine; naproxen; leflunomide; methylprednisolone acetate;indomethacin; hydroxychloroquine sulfate; sulindac; prednisone;betamethasone diprop augmented; infliximab; methotrexate; folate;triamcinolone acetonide; diclofenac; dimethylsulfoxide; piroxicam;diclofenac sodium; ketoprofen; meloxicam; prednisone;methylprednisolone; nabumetone; tolmetin sodium; calcipotriene;cyclosporine; diclofenac; sodium/misoprostol; fluocinonide; glucosaminesulfate; gold sodium thiomalate; hydrocodone; bitartrate/apap;ibuprofen; risedronate sodium; sulfadiazine; thioguanine; valdecoxib;alefacept; and efalizumab.

In one embodiment the TNFα inhibitor is administered following aninitial procedure for treating coronary heart disease in the multiplevariable dose regimen of the invention. Examples of such proceduresinclude, but are not limited to coronary artery bypass grafting (CABO)and Percutaneous transluminal coronary balloon angioplasty (PTCA) orangioplasty. In one embodiment, the TNFα inhibitor is administered inorder to prevent stenosis from re-occurring. In another embodiment ofthe invention, the TNFα inhibitor is administered in order to prevent ortreat restenosis. The invention also provides a method of treatment,wherein the TNFα inhibitor is administered prior to, in conjunctionwith, or following the insertion of a stent in the artery of a subjectreceiving a procedure for treating coronary heart disease. In oneembodiment the stem is administered following CABG or PTCA.

A wide variety of stent grafts may be utilized within the context of thepresent invention, depending on the site and nature of treatmentdesired. Stent grafts may be, for example, bifurcated or tube grafts,cylindrical or tapered, self-expandable or balloon-expandable, unibody,or, modular. Moreover, the stent graft may be adapted to release thedrug at only the distal ends, or along the entire body of the stentgraft. The TNFα inhibitor of the invention can also be administered on astent. In one embodiment, the TNFα antibody, including, for example,adalimumab/D2E7/HUMIRA® is administered by a drug-eluting stent.

The TNFα antibody can be administered in combination with an additionaltherapeutic agent to treat restenosis. Examples of agents which can beused to treat or prevent restenosis include sirolimus, paclitaxel,everolimus, tacrolimus, ABT-578, and acetaminophen.

The TNFα antibody of the invention can be administered in combinationwith an additional therapeutic agent to treat myocardial infarction.Examples of agents which can be used to treat or prevent myocardialinfarction include aspirin, nitroglycerin, metoprolol tartrate,enoxaparin sodium, heparin sodium, clopidogrel bisulfate, carvedilol,atenolol, morphine sulfate, metoprolol succinate, warfarin sodium,lisinopril, isosorbide mononitrate, digoxin, furosemide, simvastatin,ramipril, tenecteplase, enalapril maleate, torsemide, retavase, losartanpotassium, quinapril hcl/mag carb, bumetanide, alteplase, enalaprilat,amiodarone hydrochloride, tirofiban hcl m-hydrate, diltiazemhydrochloride, captopril, irbesartan, valsartan, propranololhydrochloride, fosinopril sodium, lidocaine hydrochloride, eptifibatide,cefazolin sodium, atropine sulfate, aminocaproic acid, spironolactone,interferon, sotalol hydrochloride, potassium chloride, docusate sodium,dobutamine hel, alprazolam, pravastatin sodium, atorvastatin calcium,midazolam hydrochloride, meperidine hydrochloride, isosorbide dinitrate,epinephrine, dopamine hydrochloride, bivalirudin, rosuvastatin,ezetimibe/simvastatin, avasimibe, abciximab, and cariporide.

The TNFα antibody of the invention can be administered in combinationwith an additional therapeutic agent to treat angina. Examples of agentswhich can be used to treat or prevent angina include: aspirin;nitroglycerin; isosorbide mononitrate; atenolol; metoprolol succinate;metoprolol tartrate; amlodipine besylate; digoxin; dilitiazemhydrochloride; isosorbide dinitrate; clopidogrel bisulfate; nifedipine;atorvastatin calcium; potassium chloride; simvastatin; verapamil hcl;furosemide; propranolol hel; carvedilo; lisinopril; sprionolactone;hydrochlorothiazide; enalapril maleate; madolol; ramipril; enoxaparinsodium; heparin sodium; valsartan; sotalol hydrochloride; fenofibrate;ezetimibe; bumetanide; losartan potassium,lisinopril/hydrochlorothiazide; felodipine; captopril; and bisoprololfumarate.

In one embodiment of the invention, a TNFα antibody is administered incombination with an agent which is commonly used to treat hepatitis Cvirus. Examples of such agents include Interferon-alpha-2a,Interferon-alpha-2b, Interferon-alpha con1, Interfero-alpha-n1,Pegylated interferon-alpha-2a, Pegylated interferon-alpha-2b, Ribavirin.Peginterferon alfa-2b and ribavirin, Ursodeoxycholic Acid, OlycyrrhizicAcid, Thymalfasin, Maxamine, and VX-497.

The TNFα antibody may be administered in combination with topicalcorticosteroids, vitamin D analogs, and topical or oral retinoids, orcombinations thereof, for the treatment of psoriasis. In addition, theTNFα antibody may be administered in combination with one of thefollowing agents for the treatment of psoriasis; small moleculeinhibitor of KDR (ABT-123), small molecule inhibitor of Tie-2,calcipotriene, clobetasol propionate, triamcinolone acetonide,halobetasol propionate, tazarotene, methotrexate, fluocinonide,betamethasone diprop augmented, fluocinolone, acetonide, acitretin, tarshampoo, betamethasone valerate, mometasone furoate, ketoconazole,pramoxine/fluocinolone, hydrocortisone valerate, flurandrenolide, urea,betamethasone, clobetasol propionate/emoll, fluticasone propionate,azithromycin, hydrocortisone, moisturizing formula, folic acid,desonide, coal tar, diflorasone diacetate, etanercept, folate, lacticacid, methoxsalen, bc/bismuth subgal/znox/resor, methylprednisoloneacetate, prednisone, sunscreen, salicylic acid, halcinonide, anthralin,clocortolone pivalate, coal extract, coal tar/salicylic acid, coaltar/salicylic acid/sulfur, desoximetasone, diazepam, emollient,pimecrolimus emollient, fluocinonide/emollient, mineral oil/castoroil/na lact, mineral oil/peanut oil, petroleum/isopropyl myristate,psoralen, salicylic acid, soap/tribromsalan, thimerosal/boric acid,celecoxib, infliximab, alefacept, efalizumab, tacrolimus, pimecrolimus,PUVA, UVB and other phototherapy, and sulfasalazine.

An antibody, antibody portion, may be used in combination with otheragents to treat skin conditions. For example, an antibody, antibodyportion, or other TNFα inhibitor of the invention is combined with PUVAtherapy. PUVA is a combination of psoralen (P) and long-wave ultravioletradiation (UVA) that is used to treat many different skin conditions.The antibodies, antibody portions, or other TNFα inhibitors of theinvention can also be combined with pimecrolimus. In another embodiment,the antibodies of the invention are used to treat psoriasis, wherein theantibodies are administered in combination with tacrolimus. In a furtherembodiment, tacrolimus and TNFα inhibitors are administered incombination with methotrexate and/or cyclosporine. In still anotherembodiment, the TNFα inhibitor of the invention is administered withexcimer laser treatment for treating psoriasis.

Nonlimiting examples of other therapeutic agents with which a TNFαantibody can be combined to treat a skin or nail disorder include UVAand UVB phototherapy. Other nonlimiting examples which can be used incombination with a TNFα inhibitor include anti-IL-12 and anti-IL-18therapeutic agents, including antibodies.

In one embodiment, the TNFα antibody may be administered in combinationwith an additional therapeutic agent in the treatment of Behcet'sdisease. Additional therapeutic agents which can be used to treatBehcet's disease include, but are not limited to, prednisone,cyclophosphamide (Cytoxan), Azathioprine (also called imuran,methotrexate, timethoprim/sulfamethoxazole (also called bactrim orseptra) and folic acid.

Any one of the above-mentioned therapeutic agents, alone or incombination therewith, can be administered to a subject suffering from aTNFα-related disorder in which TNFα is detrimental, in combination withthe TNFα antibody using a multiple variable dose treatment regimen ofthe invention. In one embodiment, any one of the above-mentionedtherapeutic agents, alone or in combination therewith, can beadministered to a subject suffering from rheumatoid arthritis inaddition to a TNFα antibody to treat a TNFα-related disorder. It shouldbe understood that the additional therapeutic agents can be used incombination therapy as described above, but also may be used in otherindications described herein wherein a beneficial effect is desired.

This invention is further illustrated by the following examples whichshould not be construed as limiting. The contents of all references,patents and published patent applications cited throughout thisapplication are incorporated herein by reference

EXAMPLES Example 1 Purification Procedure for Adalimumab

In this example, a purification process for purifying a mixture ofadalimumab and host cell proteins (HCPs) was devised, which process isreferred to as process A. In process A, the adalimumab-HCP mixture wasnot subjected to a protein A chromatography step. The first column usedin process A was a cation exchange resin. Fractogel S, to whichadalimumab bound while HCP flowed through. Adalimumab was then elutedfrom the Fractogel S column in a first eluate. Next, the first eluatewas subjected to pH viral inactivation to obtain a virally inactivatedpreparation. Next, the virally inactivated preparation was applied to ananion ion exchange resin, a Q sepharose column, to which adalimumab doesnot bind, to thereby obtain a first flow through. The first flow wasthen applied to a hydrophobic interaction column, a phenyl sepharosecolumn, to which adalimumab binds and HCP flows through, to therebyobtain a second eluate. Further processing and packaging of the secondeluate was performed to obtain the final bottled product.

In more detail, process A comprises the following steps:

Step 1: Fractogel S column, 100×20 cm (157 L), v=175 cm/hr, Load≦30 gprotein/L resin per cycle, equilibrated with 20 mM sodium phosphate, 25mM sodium chloride. After loading of adalimumab, the column was washedonce with equilibration buffer and eluted with an elution buffercomprising 20 mM sodium phosphate, 150 mM sodium chloride to obtain thefirst eluate;

Step 2: Delipid filtration;

Step 3: Ultrafiltration;

Step 4; pH inactivation at pH 3.5 for 1 hour; after inactivation wascomplete, pH was adjusted to 6.8 to 7.5, the filter train was washedwith two volumes of 50 nM trolamine;

Step 5: Q Sepharose F column, 60×30 cm (85 L), v=150 cm/hr, Load≦40 gprotein/L resin per cycle, equilibrated an equilibration buffercomprising 25 mM trolamine, 40 mM sodium chloride, pH-7.6; flow throughobtained;

Step 6: Phenyl Sepharose HP column, 80×15 cm (75 L), v=75 cm/hr (elute37.5 cm/hr), Load 20-40 g protein/I, resin per cycle, equilibrated withan equilibration buffer comprising 20 mM sodium phosphate, 1.1 M(NH₄)₂SO₄, pH 7, washed once with equilibration buffer and eluted byperforming a salt step-gradient to 11 mM sodium phosphate, 0.625 M(NH₄)₂SO₄, pH 7.0, to thereby obtain a second eluate, with fractionationof product peak if load≦35 g protein/L resin;

Step 7: Viral filtration;

Step 8: Final Ultrafiltration/Diafiltration;

Step 9: Final Bottling.

Further details of process A are also described in Example 2 below.

Example 2 Purification of Adalimumab to Improve Yield and DecreaseImpurities

Modifications were introduced to the capture and fine purificationoperations in the manufacturing process of the antibody adalimumab,namely process A described in Example 1 above. The modified process isreferred to herein as “process B”, and includes the following overallsteps: The starting material was the mixture obtained from thefermentation process using the Chinese Hamster Ovary (CHO) cellexpression system. The mixture was first separated using cation exchangechromatography, i.e., a Fractogel S column, where adalimumab wascaptured on the column (referred to as “capture”). The load on theFractogel S column was increased due to displacement An improved methodof washing the Fractogel S column was used to decrease the amount ofhost cell protein (HCP). The Fractogel S column with bound adalimumabwas washed with a plurality of washes, including an intermediate washwhich was a higher conductivity wash comprising 45% elution buffer and55% water for injection (WI).

Following capture and washing, adalimumab was eluted from the FractogelS column and the eluate subjected to anion exchange chromatography,i.e., a Q Sepharose column. Prior to running the first adalimumab eluateover the anion exchange column, the eluate was virally inactivated usingan improved method based on pH and conductivity. The adalimumabpreparation was collected in the flow-through of the anion column, andwas subsequently separated further according to hydrophobic interactionchromatography, i.e., phenyl sepharose column. The eluate from thephenyl sepharose column was the further processed for viral filtration,final ultrafiltration, and final bottling according to standard methodsin the art.

Process B is an improved purification method for achieving an antibodypreparation having a reduced level of HCP and procathepsin L. Theprocesses described herein were performed using a 6000 L volume,however, it should be noted that the modifications described in processB may be used with any volume. A comparison between the modifications ofprocess A versus process B is provided in Table 5 (modifications inprocess B are highlighted in bold):

TABLE 5 Comparison of process A and process B Unit operation Process AProcess B Fractogel S column 100 × 20 cm (157 L) 100 × 20 cm (157 L) v =175 cm/hr v = 175 cm/hr Load ≦30 g protein/L per cycle Load ≦35 gprotein/L per cycle Wash 1 = equilibrium buffer Wash 1 = equilibriumbuffer Elution = elution buffer Wash 2 = 45% elution buffer: 55% WFIElution = elution buffer Delipid filtration There are no changes to thisprocessing step. Ultrafiltration There are no changes to this processingstep. pH Inactivation After inactivation complete, After inactivationcomplete, adjust pH to 6.8-7.5 adjust pH to 7.8-8.2 Wash filter trainwith 2 volumes Wash filter train with of 50 mM trolamine approximately2.5 volumes of WFI to achieve conductivity in the range of 3.9-5.2 mS/cmQ Sepharose FF column 60 × 30 cm (85 L) 60 × 30 cm (85 L) v = 150 cm/hrv = 150 cm/hr Load ≦40 g protein/L resin per Load ≦40 g protein/L resinper cycle cycle Phenyl Sepharose HP column 80 × 15 cm (75 L) 80 × 15 cm(75 L) v = 75 cm/hr (elute 37.5 cm/hr) v = 75 cm/hr (elute 37.5 cm/hr)Load 20-40 g protein/L resin per Load 20-40 g protein/L resin per cyclewith fractionation of cycle with fractionation of product peak if load≦35 g/L product peak if load ≦35 g/L resin resin Viral Filtration Thereare no changes to this processing step. Final untrafiltration/ There areno changes to this processing step. diafiltration Final bottling Thereare no changes to this processing step.

The modifications to the various steps in process B are described inmore detail below:

Cation Chromatography

The primary recovery and capture operations of process B comprise depthfiltration, Fractogel SO₃ ⁻ cation exchange chromatography (FractogelS), the latter of which serves to capture adalimumab from the clarifiedharvest and reduce process-related impurities (e.g., CHO host cell andmedium impurities). A 100 cm diameter×20 cm long column (bed volume 157L) was used for this operation. The column was packed with Fractogel Sresin (EM Industries, Hawthorne, N.Y.) and the asymmetry and Height ofan Equivalent Theoretical Plate (HETP) are measured to determine thequality of the packing. The column was then sanitized with 1.0 M NaOHfor 1 hour, and stored in 0.1 M NaOH until ready for use.

Cation exchange chromatography can be affected by protein loading, ionicstrength (controlled by filtered harvest dilution), pH and linearvelocity. Protein loading can affect selectivity, resolution (purity)and yield. Ionic strength (controlled by load dilution) and pH of theload sample can affect binding capacity, selectivity, resolution andyield. Linear velocity may affect mass transport properties, potentiallyresulting in decreased binding and resolution at very high flow ratesand axial dispersion at very low flow rates.

The maximum load to the Fractogel S column was increased to 35 g proteinper liter resin. The cation column was equilibrated with 20 mM sodiumphosphate, 25 mM NaCl, pH 7. Following equilibration, the column isloaded with ≦35 g protein/L resin of diluted depth filtrate. One partdepth filtrate was diluted with approximately 1.3 parts of water toreduce the conductivity to approximately 6.1 mS/ct. The column was thenwashed to baseline with equilibration buffer followed by a wash with 9mM sodium phosphate, 68 mM NaCl, pH 7 (equivalent to 45% elution buffer,55% WFI). The product was eluted from the column in a single fractionwith 20 mM sodium phosphate, mM NaCl, pH (elution buffer). The productpool is collected from 10% full-scale deflection of the product peakA₂₈₀ on both the leading and trailing edges The column was cycled asnecessary to process the crude adalimumab. The Fractogel S eluates fromeach column cycle were pooled into the same collection tank. Betweeneach cycle, the column was regenerated with 25 mM sodium phosphate, 1.0M NaCl, pH 7.

Studies performed at laboratory scale demonstrated that efficientrecovery of product and reduction in HCP can be achieved at higher loadranges than the previously established Acceptable Operating Range (AOR)of 15 to 30 g protein/I resin. Analysis of adalimumab breakthroughversus column load indicates that the calculated 5%, breakthrough occursat 38 g/L resin at pH 7. Therefore, a revised AOR of ≦35 g protein/Lresin was established for the Fractogel S chromatography step. In sum,the load limit was also increased to 35 grams protein per liter of resinto increase process capacity.

In another set of experiments with the Fractogel resin, the effect of pHon adalimumab breakthrough versus column load was examined. Inparticular, a product breakthrough curve was used to determine the resindynamic binding capacity under defined loading conditions. Table 6 belowsummarizes the recovery data for the Fractogel loading capacity study atthe previously described pH 7 conditions. Recovery percentage at 10 g/Lwas normalized to 100%.

TABLE 6 Recovery data for Fractogel loading capacity study at pH 7Loading capacity AYF16G AYF17G Average (g/L) recovery (%) recovery (%)recovery (%) 10 100 100 100 15 99 99 99 20 98 98 98 25 99 97 98 30 98 9697 40 95 95 95 50 93 88 91 Fractogel stop yield ≧50%, BR-068.

The results at pH 7 show greater than 90% adalimumab recovery wasobserved for loading conditions of less than 50 g adalimumab per literof Fractogel resin. The adalimumab breakthrough was plotted versusloading capacity to generate a theoretical breakthrough curve. At pH 7,the theoretical 10% breakthrough was found to be at 54 g adalimumab perliter of Fractogel resin. Also at pH7, the theoretical 5% breakthroughwas found to be at 38 g adalimumab per liter of Fractogel resin,confirming the results described above.

A similar study was carried out as described above except that the loadand the first wash pH conditions were adjusted to pH 5. The cationcolumn was equilibrated with 24 mM citric acid and 51 mM sodiumphosphate, dibasic, pH 5. Following equilibration, the column was loadedwith up to 80 g protein/L resin of diluted depth filtrate. The cellculture harvest was pH adjusted to 5.0 with 3M acidic acid prior to thedepth filtration. One part depth filtrate was diluted with approximatelythe same volume of water to reduce the conductivity to approximately 8to 10 mS/cm. Again, the adalimumab breakthrough was plotted versusloading capacity to generate a theoretical breakthrough curve. Under thestudied conditions, the theoretical 10% breakthrough was found to be atapproximately 74 g adalimumab per liter of Fractogel resin. Thetheoretical 5% breakthrough was found to be at approximately 73 gadalimumab per liter of Fractogel resin. Due to the character of thecation exchange of the resin, lowering the pH of the chromatographyconditions significantly increased adalimumab dynamic binding capacity.Comparing the breakthrough curves at pH 5 and pH 7, the binding betweenadalimumab molecule and the Fractogel resin was observed to be muchstronger at lower pH. It was also found that with the higher loadingdynamic capacity at pH 5, better HCP clearance was achieved. Table 7below summarizes the data for the Fractogel loading capacity study vs.the HCP present in the eluate at the newly tested pH 5 conditions. Thedata clearly indicate that under the tested conditions, HCP displacementby adalimumab had occurred.

TABLE 7 HCP Present in Fractogel Eluate at Different Loading Capacitiesat pH 5 HCP (ng Loading capacity HCP/mg (g/L) adalimumab) 15 6102 206782 25 5767 30 5167 40 3983 60 3207

Since the analysis of adalimumab breakthrough versus column load at pH 5indicated that the calculated 5% breakthrough occurs at 73 g/L resin, arevised AOR of ≦ 70 g protein/L resin can be established for theFractogel S chromatography step at pH 5. In sum, the load limit, whichpreviously was increased to 35 grants protein per liter of resin at pH 7(as described above), can be further increased to 70 grams protein perliter of resin by lowering of the pH to 5.

Intermediate Wash

To further reduce the amount of impurities in the adalimumabpreparation, an intermediate wash step was performed prior to adalimumabelution front the cation column (see Table 8 below). This additionalwash was adjusted relative to the conductivity of the elution buffer,and helped to improve clearance of HCPs. The insertion of anintermediate wash step prior to elution reduced the amount of HCP elutedwith adalimumab by over 60% compared to process A. Parametersinvestigated included the blend of elution buffer with water used in thewash (% elution buffer), conductivity, pH, wash volume, flow rate andresin age. The optimum wash consists of a blend of 45% elution buffer(20 mM sodium phosphate, 150 mM sodium chloride, pH 7) and 55% water.Table 8 presents data comparing the level of HCP in the Fractogel Seluate with and without the additional wash. Fractogel S eluate sampleswere assayed for HCP and compared with HCP levels in the eluate from apilot-scale Fractogel S process, which incorporated a higher load andthe wash step. The pilot-scale data indicates that the addition of thesecond wash step significantly improves the clearance of HCP by theFractogel S step.

TABLE 8 HCP levels in Fractogel S eluate with and without pre-elutionwash step at laboratory scale Column Load g protein/L Step yield HCP(ng/mg Sample resin (%) adalimumab) Lot A no 2^(nd) wash^(a) 25 96 19410Lot B no 2^(nd) wash^(a) 25 96 22992 Lot C no 2^(nd) wash^(a) 25 9321931 Lot D no 2^(nd) wash^(a) 25 97 20037 6000 L load^(b),pilot-scale^(c) 30 95 4914 with 2^(nd) wash step ^(a)Fractogel S eluatesample was taken from the indicated 6000 L lot and analyzed for HCPcontent ^(b)Load consisted of a blend of filtered harvest from variouslots ^(c)Pilot-scale column size is 10 (D) × 21 (L) cm; 2^(nd) washbuffer: 45% elution buffer (20 mM sodium phosphate, 150 mM sodiumchloride, pH 7.2), 55% water for injection.

Typical elution profiles for the Fractogel S chromatography step foreach process are provided in FIG. 1. Process B includes theabove-mentioned intermediate additional wash step prior to elution, thusthe leading edge of the elution peak is sharper with less early-elutingspecies detected than that of the previous process. In sum, anintermediate wash step, just prior to the elution of adalimumab, wasintroduced to the Fractogel S step to improve clearance ofprocess-related impurities, such as HCPs.

Viral Inactivation

The low pH inactivation step of process B provides a margin of safety byinactivating potential undetected enveloped viruses that may be presentin the delipid filtrate. The viral-inactivated pool is subsequentlypH-neutralized and filtered to remove particulates and minimizebioburden. The quality of the adalimumab during low pit virusinactivation may be affected by pH and the duration of the low pHincubation. Virus inactivation is dependent on these same parameters,and it may be affected by the protein concentration, which may reduceinactivation at high concentrations. The minimum incubation time at lowpH was increased from 15 minutes to 60 minutes. Analysis ofmanufacturing samples taken before and after the low pH step confirmedthat adalimumab can be safely held at pH 3.5 for 1 hour withoutcompromising its ability to protect murine L929 cells against thecytotoxic effects of tumor necrosis factor (TNF).

Following inactivation, the pH and conductivity of the viral-inactivatedeluate were adjusted in accordance with the equilibration buffer of thefollowing column, e.g., Q Sepharose column. The pH was adjusted to7.8-8.2, with a target pH of 8.0. In sum, the pH and conductivity of theviral-inactivated pool, which serves as the Q Sepharose FF load, wasadjusted to match to the pH and conductivity of the Q Sepharoseequilibration buffer.

Anion Chromatography

The anionic column, i.e., Q Sepharose, step serves to reduceprocess-related impurities such as HCP, specifically includingprocathepsin L, as well as DNA and insulin. A 60 cm diameter×30 cm longcolumn (bed volume 85 L) was used for Q Sepharose FF chromatography. Thecolumn was packed with Q Sepharose FF resin (Amersham Pharmacia,Piscataway, N.J.) and asymmetry and HETP were measured to determine thequality of the packing. The column was then sanitized with 1.0 M NaOHfor 1 hour, and stored in 25 mM sodium phosphate, 20% isopropanol untilready for use.

Equilibration of the resin was accomplished with 25 mM trolamine, 40 mMNaCl, pH 8 (equilibration buffer). The maximum protein loading for thisstep was ≦40 g protein/L of resin per cycle. Process-related impuritiesadsorbed to the resin, and adalimumab flowed through the column. Thediluted, filtered, virus-inactivated material was typically processed intwo cycles of approximately equal amounts; additional cycles may berequired to process all available material. Loading and elution wereperformed at 150 cm/hr, and the column flow-through is collected whenthe A₂₈₀ rises above 2% full scale. The column was then washed withequilibration buffer and the wash was collected until the A₂₈₀ returnsto 5% full scale. The wash is pooled with the low-through and isdesignated Q Sepharose FTW. Between cycles, the column was regeneratedwith 25 mM sodium phosphate, 1.0 M NaCl, pH 7, and then equilibratedwith equilibration buffer.

Anion exchange chromatography operated in flow-through mode can beaffected by protein loading, ionic strength (conductivity, which may becontrolled by dilution of the low pH inactivation filtrate), pH andlinear velocity. Protein loading can affect selectivity and yield. Ionicstrength and pH of the load sample can affect binding capacity andselectivity. Linear velocity may affect mass transport properties,potentially resulting in decreased binding of process related impuritiesat very high flow rates and axial dispersion at very low flow rates. Newload conductivity and pH ranges have been established based onlaboratory studies

Laboratory studies indicated that reduction of HCP by the Q Sepharose FFstep could be enhanced by alterations to the loading conditions.Parameters investigated included the load pH, conductivity and grains ofprotein loaded per L of resin. Adjustment of the load conductivity andpH to match that of the column equilibration buffer (5 mS/cm, pH 8), andlimiting the load≦40 g adalimumab/L resin result in improved clearanceof HCP and procathepsin L. Table 9 presents laboratory-scale datashowing the reduction in HCP under process A (pH 7.7, conductivity 6.65mS/cm) and the improved process conditions of pH 8 and conductivity of 5mS/cm of process B. Limiting the load on the Q Sepharose column to 40g/L of resin provides a four-fold improvement in clearance of HCP andthe additional modifications to the pH and conductivity of the loadyield a three-fold further improvement in HCP reduction.

TABLE 9 HCP reduction under varying Q Sepharose FF load conditions LoadHCP Flow-through Load amount (ng/mg HCP (ng/mg Fold reduction Gprotein/L resin Load conditions adalimumab) adalimumab) in HCP 80 pH7.7, 6.65 mS/cm 726 452 1.6 40 pH 7.7, 6.65 mS/cm 726 114 6.4 40 pH 8.1,5.08 mS/cm 726 37.6 19.3

The HCP-reduced flowthrough comprising adalimumab obtained from the ionexchange column was subsequently used in hydrophobic interactionchromatography.

Hydrophobic Interaction Chromatography

The objective of the Phenyl Sepharose HP chromatography column was tofurther reduce process-related and product-related impurities such ashost cell proteins and aggregates, respectively. An 80 cm diameter×15 cmlong column (bed volume L) was used for this operation. The column waspacked with Phenyl Sepharose HP resin (Amersham Pharmacia, Piscataway,N.J.) and asymmetry and HETP were measured to determine the quality ofthe packing. The column was then sanitized with 1.0 M NaOH for 1 hour,and stored in 25 mM Na Phosphate, 20% isopropanol until ready for use.

Equilibration of the resin was accomplished with 20 mM Sodium phosphate.1.1 M (NH₄)₂SO₄, pH 7.0 (equilibration buffer). The protein loading forthis step was 20 to 40 g protein per L of resin, and two or threechromatography cycles were required to process the entire quantity ofavailable material. The column operated at a linear velocity of 75cm/hr. The Q Sepharose flowthrough was diluted with an equal volume of40 mM sodium phosphate, 2.2 M (NH₄)₂SO₄, pH 7.0. Following loading thecolumn was washed with 20 mM sodium phosphate. 1.1 M (NH₄)₂SO₄, pH 7.0.The product was eluted by performing a salt step-gradient to 11 mMsodium phosphate, 0.625 M (NH₄)₂SO₄, pH 7.0. Product was collected asthe absorbance rises above 50% UV full scale and continued untilabsorbance decreases to less than 20% UV full scale as the peak tails.

The process modifications to the Fractogel S and Q Sepharose FFchromatography steps significantly reduced the burden of process-relatedimpurity reduction placed upon the Phenyl Sepharose HP step. As aconsequence of the changes, the major function of the Phenyl SepharoseHP step was the removal of adalimumab aggregates.

Process A required that at column loads of 35 g protein/L, resin orhigher, product was collected as the U V absorbance rises above 50% fullscale deflection and continues until absorbance decreases to <20% fullscale. At column loadings below 35 g protein/L, resin, the first 0.15column volume of the eluate peak was excluded from the collected pool toimprove HCP clearance at this step. The incorporation of themodifications at the previous chromatography steps in process Balleviated the need for the peak exclusion at loads below 35 g protein/Lresin since the incoming HCP load was significantly reduced. Thereduction in the HCP load allowed expansion of the load range withoutfractionation. The effect of this change permits processing of allmaterial from each fermentation by the recovery process without PhenylSepharose HP peak cutting

The linear flow rate for Phenyl Sepharose operation was investigated atlaboratory scale. The adalimumab load was held constant and flow ratesof 25 to 125 cm/hr were examined. The flow rate did affect productrecovery but had no impact on product quality as assessed by SEC (%monomer) and clearance of HCP (Table 10), justifying the broader rangeof 25 to 125 cm/hr. The target flow rates for the Phenyl Sepharosemanufacturing operation remain as previously established at 75 cm/hr and37.5 cm/hr fir the elution phase.

TABLE 10 Phenyl sepharose flow rate evalution Flow rate Load (gprotein/L % % % HCP (cm/hr) resin) Recovery^(a) Monomer^(b) clearance 2532.5 69 99.98 92.1 75 32.5 84 99.98 92.2 125  32.5 84 99.98 91.8^(a)Phenyl Sepharose step yield action limit: ≧48% ^(b)Phenyl Sepharosestep SEC action limit: ≧98% monomer

The acceptable operating ranges for Phenyl Sepharose HP chromatographywere investigated. Hydrophobic interaction chromatography can beaffected by protein loading, ionic strength (conductivity), and linearvelocity. Protein loading can affect selectivity and yield. Ionicstrength of the load sample can affect binding capacity, selectivity andresolution. Linear velocity may affect mass transport properties,potentially resulting in decreased resolution of process relatedimpurities at very high flow rates and axial dispersion at very low flowrates. The linear flow rate range is expanded to 25 to 125 cm/hr. Theother acceptable operating ranges for Phenyl Sepharose chromatographyare unchanged from those previously established for the 6000 L processand are listed in Table 10.

Comparable performance of the fine purification operations in bothprocesses was demonstrated. Changes introduced as part of improvedprocess B include: adjustment of the pH and conductivity of theviral-inactivated pool, which serves as the Q Sepharose FF load, tomatch the Q Sepharose equilibration buffer, limiting the Q Sepharoseload to less than 40 g protein per liter resin, and elimination of therequirement to fractionate the Phenyl Sepharose eluate at loads of lessthan 35 g protein per liter resin. The quality of intermediates, asdetermined by SEC and WCX-10 assays, were comparable between the twoprocesses.

Typical elution profiles for the Q Sepharose PP and Phenyl Sepharose HPchromatography steps for each process are provided in FIGS. 2 and 3,respectively. The Q Sepharose flow-through comprising adalimumab and wascollected. The load volume amounts for process B were higher than theprevious process, due to the greater loads at the previouschromatography step (Fractogel S) and increased dilution volume;therefore the total flow-through volume is correspondingly greater.

In sum, a requirement to fractionate the Phenyl Sepharose eluate forloads less than 35 g protein per liter resin was eliminated due toimprovements in impurity clearance resulting from the changes in theFractogel S and Q Sepharose operations. In addition, the linear flowrate range was expanded to 25 to 125 cm/hr.

Reduction in HCP

Process B included modifications to the Fractogel S and Q Sepharosechromatography steps which were implemented to improve control ofprocess-related impurities such as host cell protein (HCP) and,specifically, procathepsin L. A study was undertaken to assess theimpact of process B on the removal of these impurities. The capacity ofthe Fractogel S, Q Sepharose FF and Phenyl Sepharose HP columns toremove CHO host cell proteins was evaluated at manufacturing scale. Hostcell protein levels were determined by HCP ELISA (see Example 3) anddata are expressed in ng HCP/mg adalimumab.

Representative samples were taken during process B and assayed for HCP.The results are presented in Table 11. Changes to the chromatographysteps represent more rigorous chromatographic conditions which would beexpected to improve the T HCP clearance. Delipid filtration resultsreported are those from process A. The delipid filtration step wasunchanged process B, therefore the HCP reduction factor achieved at thisstep is included in the overall performance of process B. On average,process B is able to remove greater than 4.35 log₁₀ of HCP. BothFractogel S chromatography and Q Sepharose FF chromatography clearedmore than 1 log₁₀ HCP, and the depth filtration step also cleared morethen 1 log₁₀. Additional HCP was removed by the Phenyl Sepharose column,however, the clearance value was not calculable because both the loadand eluate HCP levels were below the level of quantitation. The drugsubstance produced by process B exhibited HCP levels below the limit ofquantitation (LOQ) for the three validation lots.

TABLE 11 Host cell protein clearance Chromatography HCP in HCP out Log₁₀reduction step (ng/mg ada) (ng/mg ada) factor Fractogel SO₃— 1.71 column(average)^(a) Lot D 1,035,101 18,199 1.75 Lot E 747,748 16,079 1.67 LotF 1,350,632 26,772 1.70 Delipid Filtration 1.58 (average)^(b) Lot 18,1741,466 1.11 Lot H 34,369 805 1.63 Lot I 38,453 570 1.83 Lot J 25,774 4661.74 Q Seph/ FF column 1.07 (average)^(a) Lot D 269.98 Cycle A: 28.410.98 Cycle B: 30.54 0.95 Lot E 313.44 Cycle A: 28.94 1.03 Cycle B: 29.821.02 Lot F 391.96 Cycle A: 22.51 1.24 Cycle B: 26.52 1.17 Phenyl Seph.HP N/A column (average)^(a) Lot D <40.44 <9.08 N/A Lot E <43.56 <8.65N/A Lot F <44.65 <9.22 N/A Total Clearance^(c) 4.35 ^(a)Data fromprocess B ^(b)Data from process A ^(c)Log 10 reduction factors less than1 are not included in the overall clearance calculation

Overall improvements in HCP and procathepsin L levels are also shown inTables 12 and 13, respectively, where process R shamed significantdecreases in both levels in comparison to process A.

Procathepsin L Process Mapping

Process intermediate samples were taken at several steps and analyzedfor fluorescence generated by activation of procathepsin L to cathepsinL. Results are shown in Table 14 below for process B and process Asamples. The Fractogel S load and Phenyl Sepharose load and eluatesamples could not be evaluated due to interference with the method. TheQ Sepharose FF chromatography step has the capability of removinggreater than 90% of the detectable enzyme in the load. The Q Sepharoseflow-through and wash (FTW) from the improved process containsapproximately 50% less activatable procathepsin L than the Q SepharoseFTW From the 6000 L previous process. Reductions also occur between theFractogel S and the Q Sepharose steps during which the delipidfiltration, concentration by ultrafiltration, low pH viral inactivationand depth filtration operations are performed.

TABLE 14 Procathepsin L mapping of the process A and process B Process B(RFU/s/mg) Average ± Reduction Sample Lot S Lot U Lot T SD Factor^(a)Fractogel Eluate Pool 46 59 57  54 ± 7 N/A Q Sepharose load 32 31 39  34± 4 1.6 Q Sepharose FTW 2.2 3.6 2.3 2.7 ± 0.8 13.4 Drug substance 2.63.0 2.7 2.8 ± 0.2 None Process A Average ± Reduction Lot V Lot W Lot XSD Factor Fractogel Eluate Pool 96 83 96  91 ± 7 N/A Q Sepharose load 4667 58  57 ± 10 1.6 Q Sepharose FTW 5.8 5.7 5.0 5.5 ± 0.5 10.5 Drugsubstance 4.2 3.9 3.8 4.0 ± 0.2 1.4 ^(a)The reduction factor iscalculated using pre-rounded data for each lot and the average of thethree runs is reported.The comparison of procathepsin L reduction in processes A and B isdisplayed in FIG. 4. Process B exhibits lower procathepsin L levels thanprocess A at each intermediate step, indicating that the modificationsto the Fractogel S and Q Sepharose chromatography steps improve processperformance with respect to removal of this impurity.

HCP Process Mapping

Process intermediate samples were collected from both processes A and B,and analyzed for HCP content. This study was performed in order todirectly compare the two processes for HCP reduction. The results of theHCP analysis are shown in Table 15. Significant removal of HCP occurs atthe Fractogel S and Q Sepharose steps in both processes but process Bexhibits improved HCP clearance across both of these steps. The improvedFractogel S step, which includes the second wash step prior to productelution, has a reduction factor of 96 (1.96 log₁₀) whereas the same stepin the previous process yields a reduction factor of 48 (1.67 log₁₀).Both processes exhibit a reduction factor of 50 accomplished by thedelipid filtration, performed between the Fractogel S and Q Sepharosechromatography steps. The Q Sepharose operation in process B isperformed with the load adjusted to the pH and conductivity of thecolumn equilibration buffer. The HCP reduction factor achieved by theimproved Q Sepharose step is four-fold greater than that demonstrated bythe previous process (21 vs. 5). Further reduction occurs across thePhenyl Sepharose step such that the level of HCP is below the level ofquantitation in the improved process UF/DF pool and drug substance; theprevious drug substance samples exhibit very low but measurable levelsof HCP.

TABLE 15 Host cell protein mapping of processes A and B Process B lots(ng HCP/mg adalimumab) Average ± Reduction Sample Lot D Lot E Lot F SDFactor Filtered Harvest 1,330,000 813,000 2,130,000 1,420,000 ± 661,000 N/A Fractogel Eluate Pool 12,400 19,200 15,300 15,600 ± 3370  96 QSepharose load 554 220 371  382 ± 167 50 Q Sepharose FTW 18.5 20 17 18.5± 1.5 21 Drug substance^(a) <5 <5 <5 <5 >4 Process A lots (ng HCP/mgadalimumab) Average ± Reduction Lot V Lot W Lot X SD Factor FilteredHarvest 2,030,000 2,520,000 1,870,000 2,140,000 ± 339,000  N/A FractogelEluate Pool 40,400 40,700 56,400 45,800 ± 9160  48 Q Sepharose load 5361347 1248 1040 ± 442 50 Q Sepharose FTW 98 213 283 198 ± 93 5 Drugsubstance 5 8 11  8 ± 3 24 ^(a)All improved lot samples for this stepwere below the 5 ng/mg limit of quantitation. A value of 5 ng/mg wasused to estimate the reduction factor.The comparison of HCP reduction in process B versus process A, plottedon a log₁₀ scale, is displayed in FIG. 5. Process B exhibits lower HCPlevels than process A at each intermediate step, including a 10-folddifference following the Q Sepharose step, indicating that themodifications to the Fractogel S and Q Sepharose chromatography stepsimprove process performance with respect to removal of HCPs.

Impact of the Capture and Fine Purification Operations on ProcessingCapacity

Two changes were introduced to increase the processing capacity of thecapture and fine purification operations in process B. The first was theincrease in the allowable load limit on the Fractogel S column from 30 gprotein/L resin to 35 g protein/L resin at pH 7 and from 30 g protein/L,resin to 70 g protein/L resin at pH 5. These changes allowed all of thefiltered harvest material from the bioreactor to be loaded onto theFractogel S column. The average load onto the Fractogel S column wasapproximately 9% higher in the improved process (at pH 7) than the loadin the previous process (Table 16).

The second change was the removal of the requirement to fractionate thePhenyl Sepharose product peak with loads of less than 35 g protein/Lresin. The fractionation resulted in discarding a significant portion ofthe product peak in order to adequately control host cell proteins. Thechanges implemented at the Fractogel S and Q Sepharose steps to controlhost cell proteins and procathepsin L levels rendered the fractionationof the Phenyl Sepharose peak unnecessary. This change allowed runningthree cycles of the Phenyl Sepharose column at a lower load range forprocess B resulting in a 12% increase in total load on the PhenylSepharose column compared with process A.

Table 16 compares the loads on the Fractogel S and the Phenyl Sepharosecolumns as well as the final drug substance amounts from the improvedand previous processes. The improved process exhibits an approximate 8%overall increase in adalimumab yield for the three validation batches.

TABLE 16 Comparison of Fractogel S and Phenyl Sepharose column loads anddrug substance yields in processes A and B Fractogel S Phenyl DrugProcess load^(a) Sepharose load^(a) Substance yield^(a) Process A (n =15) 7641 ± 138 5947 ± 28 5290 ± 158 Process B (n = 3) 8375 ± 293 6752 ±38 5748 ± 75  Increase in process B 9% 12% 8% ^(a)Load and yield areexpressed in grams of protein

The improved method of purifying the antibody adalimumab improvedclearance of HCP and procathepsin L (relative to process A), resultingin reduced levels in the drug substance. More specifically, in acomparison of drug substance lot release data, the following levels ofHCP and procathepsin L were determined, as described in Table 17.

TABLE 17 Comparison of HCP and procathepsin L in processes A and B Lotrelease specifi- Process Process Process Process Process Assay cationA.1 A.2 A.3 A.4 B Host cell ≦70 ng/mg 46 ± 15 6 ± 3^(b) 22 ± 19   9 ± 4<5   protein (HCP) Procath- ≦5% 18 ± 8^(a) <3.85^(c) 65 ± 23²³ <3.61^(d)<3.3^(e) epsin L ^(a)Procathepsin L specification does not apply toprocess A.1; values provided for information only. ^(b)14 of 17 lotsbelow LOQ limit of 5 ng/mg; a value of 5 ng/mg used to calculate averageand standard deviation. ^(c)LOQ ranged from 3.30 to 3.85. ^(d)LOQ rangedfrom 3.29 to 3.61. ^(e)LOQ was 3.3 (LOQ = Limit of quantitation)

Extended characterization of the drug substance produced using theprocess B was performed. Drug substance from the three validation lotswas analyzed and compared with an adalimumab reference standard, usingthe assays including amino acid analysis, circular dichroism, analyticalcentrifugation, QSTAR LC-mass spectrometry, non-reduced tryptic and LYSC peptide mapping with MS detection, free sulfhydryl assay, trypticpeptide mapping with MS detection, immunoblot, L929 bioassay, andBIAcore. All batches of drug substance manufactured by the improvedprocess met the acceptance criteria and are comparable to the referencestandard.

In sum, the performance of the process B has been demonstrated to becomparable to the process A at fermentation, capture and finepurification stages. Process B, however, exhibits improved capabilitywith regard to reduction of host cell protein and procathepsin L, aswell as an increase in capacity with regard to adalimumab yield. Drugsubstance release testing and extended characterization studies furtherdemonstrate the comparability of the adalimumab drug substance producedby process B with that produced by process A.

TABLE 12 Overall improvement of HCP levels. Sample descrip- Process A.1(3 K) Process A.2 (2 K) tion A B C D E F  1 Post 853,852 1,181,845936,390 1,238,297 991,390 1,018,529 depth filtration  2 Frac- 6,73915,772 16,286 17,528 15141 15426 togel-S eluate  3 Conc. 5980 13,95815,361 14984 12769    434* Frac- togel-S eluate G H I J K L  4 Viral2702 5074 5181 3826 3321 216 inacti- vated filtrate  5 Q Seph. 415 891562 311 157 30 FF flow through  6 Final 36 83 43 20 7 <LOQ UF/DFretentate HCP Q 6.51 5.69 9.22 12.30 21.16 7.20 reduction (fold) Sam-Sample ple descrip- Process A.3 (6 K) Process A.4 (6 K) Process B.1 (6K) ID tion M N O P Q R S T  7 Post 2,039,630 2,150,284 2,125,9862,026,000 2,517,074 1,867,919 1,333,900 813,256 depth filtration  8Frac- 35,223 31,461 46,072 40,399 40,710 56,415 12,430 19,150 togel-Seluate Y Z A.A A.B A.C A.D A.E A.F  9 Conc. 1157 NA NA 2,325 2,553 2,437NA NA Frac- togel-S eluate 10 Viral 468 527 1,563 536 1,347 1,248 554220 inacti- vated filtrate 11 Q Seph. 229 314 594 98 213 283 18.5 20 FFflow through 12 Final 23 33 48     5* 8 11 0.5 0 UF/DF retentate HCP Q2.04 1.68 2.63 5.47 6.32 4.41 reduction (fold) Sample Sample Process B.1(6 K) Process B.2 (12 K) ID description U V W X  7 Post 2,126,4491,271,211 1,261,889 1,056,935 depth filtration  8 Frac- 15,257 9,55813,130 17,427 togel-S eluate A.G  9 Conc. NA 4824 771 526 Frac- togel-Seluate 10 Viral 371 1,317 376 255 inacti- vated filtrate 11 Q Seph. 1714 3 0 FF flow through 12 Final 0 0 0 0 UF/DF retentate HCP Q reduction(fold) *lot was operated with delipid filter in the process

TABLE 13 Overall improvements in procathepsin L levels. Sam- ple SampleProcess A.1 (3 K) Process A.2 (3 K) ID description A B C D E F  1Fractogel-S 54.07 51.23 61.04 42.19 50.14 48.28 eluate  2 Conc. 45.7642.95 48.44 46.72 43.77 46.13 Fractogel-S eluate G H I J K L  3 Viral41.54 36.37 43.70 36.49 38.28 38.94 inactivated filtrate  4 Q Seph. 6.55  6.22  7.16  3.39  3.37  3.41 FF flow through  5 Phenyl  6.82 6.92  8.75  2.57  2.11  3.01 Seph FF eluate  6 Final  9.85  9.77  9.69 1.84  2.93  3.00 UF/DF retentate Cathepsin  6.34  5.85  6.10 10.7611.36 11.42 L Activity Q reduction (fold) Sam- ple Sample Process A.3 (6K) Process A.4 (6 K) Process B.1 (6 K) Process B.2 (12 K) ID descriptionM N O P Q R S T U V W X  7 Fractogel-S 98.26 77.10 106.41 97.42 84.9194.14 45.78 58.55 56.61 26.70 40.70 41.05 eluate Conc. 61.94 57.20 47.2022.20 42.80 36.53 Fractogel-S eluate Y Z A.A A.B A.C A.D A.E A.F A.G  8Viral 51.49 64.38  69.29 46.43 67.28 57.59 31.85 31.19 38.56 13.20 45.9042.32 inactivated filtrate  9 Q Seph. 10.02  9.86  10.05  5.81  5.65 4.96  2.21  3.57  2.25  0.40  1.30  0.9 FF flow through 10 Phenyl  8.48 8.79   9.21  3.29  4.28  3.48  1.00  1.30  0.85 Seph FF eluate 11 Final 6.68  8.76   8.54  3.28  3.46  3.71  2.59  3.03  2.65  1.30  0.60  1.00UF/DF retentate Cathepsin L  5.14  6.53   6.89  7.99 11.91 11.61 14.41 8.74 17.14 33.00 35.31 47.02 Activity Q reduction (fold) Cathepsin Lactivity: The unit used in red numbers is the fluroscent release ratedescribed as RFU/sec./mg D2E7

Example 3 Assay for HCP Detection

The following example describes an HCP EISA method for the determinationof the residual Host Cell Protein (HCP) concentration in adalimumab drugsubstance samples obtained from process B, described in Example 2.Enzyme Linked Immunosorbent Assay (ELISA) was used to sandwich thesample comprising the HCP antigen between two layers of specificantibodies. This was followed by the blocking of non-specific sites withCasein. The sample was then incubated during which time the antigenmolecules were captured by the first antibody (coating antibody Cygnusgoat anti-CHO (Chinese Hamster Ovary), affinity purified). A secondantibody (anti-CHO host cell protein biotinylated) was then added whichfixed to the antigen (CHO host cell proteins). Importantly, the secondantibody specific to the HCPs was produced from the cells used togenerate the antibody. Neutravidin HRP-conjugated was added which bindsto the biotinylated anti-CHO host cell protein. This was followed by theaddition of K blue substrate. The chromogenic substrate was hydrolyzedby the bound enzyme conjugated antibody, producing a blue color. Thereaction was stopped with 2 M H₃PO₄, changing color to yellow. Colorintensity was directly proportional to the amount of antigen bound tothe well. The HCP ELISA showed improvements for determining HCP levelsin an antibody preparation than standard ELISA methods.

Example 4 Cathepsin L Kinetic Assay

A kinetic assay was developed and used to quantify cathepsin L activityfor adalimumab manufacturing process intermediates of process B (seeExample 2). The weak anion exchange HPLC assay (WAX-10 HPLC) used tomeasure HCP for drug substance release testing could not be used forthis study since the variable protein content and buffer composition ofthe in-process samples may interfere with the method. The inability todirectly quantitate procathepsin L in the process intermediates led tothe development of an assay which measured the activity of cathepsin Lby a kinetic fluorescence method. The kinetic assay, i.e., a highthroughput fluorescent enzymatic method, has less interference forin-process samples than standard methods used to detect procathepsin Llevels. The kinetic assay also provides a means for examining thereliability of the process for purifying adalimumab in-process samplesdescribed in Examples 1 and 2.

This method forces the activation of the procathepsin L in the samplesto cathepsin L by addition of dextran sulfate. A fluorogenic peptidesubstrate, Z-leucine-arginine-AMC (7-amino-4-methyl coumarin), was usedto detect cathepsin L activity at excitation 380 nm and emission 460 nm.The level of fluorescence activity in the samples was determined by theslope of the fluorogenic signal generated by the cleavage of thesubstrate per second. The range of this fluorescent activity assay wasdetermined to be between 0.0144 to 1.04 RFU/sec. This activity wascorrelated to the amount of adalimumab present in the test sample; henceresults are report as RFU/sec/mg adalimumab. Optimum activationconditions to achieve the maximum fluorescent signal were developed foreach process intermediate sample using JMP software derived DOEexperiments. The recommended activation conditions for this assay aresummarized in Table 16.

Materials and Methods

Preparation of 500 mM DTT Stock Solution

7.7 grams of Ultrapure DTT (Invitrogen) was added into 90 mL of Milli-Qwater and mixed until homogenous. The solution was topped up thesolution with Milli-Q Water to a final volume of 100 mL. This 500 mM DTTstock was then aliquoted and stored at −80° C.

Preparation of the Activation Buffer (25 mM NaOAc, 5 mM DTT; 1 mM EDTApH 5.5)

3.44 grams of sodium acetate (J. T. Baker), 0.38 grams of EDTA (J. T.Baker) and 950 mL Milli-Q water were added to a proper container andmixed until completely homogenous. The pH of the buffer was adjusted to5.5 with 1 M HCl, and brought up to the final volume of 1 L in avolumetric flask. The buffer was filtered through a 0.22 μm filter andstored at 4° C. prior to use. 500 μL of DTT stock solution (500 mMdescribed above) was added to 50 mL of buffer to a final concentrationof 5 mM at the day of use.

Preparation of Dextran Sulfate+0.1% Sodium Azide Stock Solution

1 gram of dextran sulfate (EM Science) was added into 90 mL of Milli-Qwater and mixed by until homogenous. 100 μL sodium azide was added froma 1 mg/mL stock solution (J. T. Baker). The solution was topped up to afinal volume of 100 mL. This solution was then aliquoted and stored at−80° C.

Kinetic Assay Set-Up

Samples to be tested for cathepsin L activity require activation of theproenzyme (procathepsin L) to active enzyme (cathepsin L). This wasaccomplished by diluting samples in activation buffer, adding dextransulfate and incubating at 37° C. for an appropriate time (detailsdiscussed in below). After activation, samples can be stored at −80° C.and remain stable. Optimal activation conditions determined forin-process samples are shown in Table 18.

TABLE 18 Summary of refined activation conditions for in-process samplesDextran sulfate Activation Sample Dilution (μg/mL) time (hr) Fractogeleluate 700 0.035 6 Q Sepharose load 700 0.035 6 Q Sepharose FTW 70 0.03518 Phenyl eluate 200 0.035 6 Drug substance 600 0.035 6

On the day of testing, an aliquot of the test samples were removed from−80° C. and thawed in an ice bath. Once the test samples have thawed,(2×) 100 μL of each sample was loaded into a black polystyrene microtiter plate (Corning cat#3650). An aliquot of the Z-L-R-AMC FluorogenicPeptide Substrate VII (R&D Systems) was thawed while protected fromlight. The substrate was diluted 1:1350 with the acetate buffer to afinal concentration of 20 μM. 100 μL of the fluorogenic substrate wasadded to each well. The plate was then mixed for ˜1 second and incubatedat 37° C. for minutes, while protected from light. The plate was thenplaced in the fluorescent plate reader that has been set to 37° C. Theexcitation wavelength was set to 380 nm and the emission was set to 460nm. The fluorescence of each well was measured every 3 minutes for 30minutes and the rate of substrate hydrolysis was calculated. Theresults, which take into consideration the dilution factor, were thendivided by the adalimumab concentration for comparison. Results usingthis kinetic assay are described above in Example 2.

Adalimumab concentration was determined by A₂₈₀ using an extinctioncoefficient of 1.39. Adalimumab quantitation was performed on studysamples using Poros A analysis. Sample dilutions were applied to achievereadings within the standard curve. A Shimadzu HPLC system wasconfigured with a Poros A ImmunoDetection sensor cartridge (AppliedBiosystems, Foster City. Calif.). The column was maintained at ambienttemperature. The system was run at 2 mL/minute. The auto sampler traytemperature was set at 4° C. Absorbance was monitored at 280 nm. BufferA was 1×PBS; buffer B was 0.1 M acetic acid and 150 mM sodium chloride.The sample was injected and Adalimumab was eluted using 100% buffer B.

The turnover of fluorogenic peptide using Fractogel load (see firsteluate Example 2; process B) front material obtained from CHO) cellexpression of adalimumab is shown in FIG. 6. This sample was diluted to200, 50 and 20 μg/mL of adalimumab with activation buffer using 0.5μg/mL dextran sulfate, and incubated at 37° C. for 16 hours. This lot at50 and 20 μg/mL showed linear responses. The R² values are ≧0.99.However, the lot at 200 μg/mL shows nonlinear substrate turnover towardsthe end of the 30 minutes measurement time, resulting in a lower R²value of 0.91. Therefore, careful sample dilution is critical tomaintain linear hydrolysis rates.

Assays were also performed to confirm that the kinetic assay usingcathepsin activity to determine the level of procathepsin A werecompliant with ICH guidelines, including precision analysis, includingrepeatability precision. Furthermore, it was determined that the type ofcontainer, e.g., glass and polypropylene vials influences of cathepsin Lactivity. The results suggest that higher levels of cathepsin L areachieved when incubating in a polypropylene container as opposed to aglass container. In both cases, the addition of 0.5 μg/mL dextransulfate was required for procathepsin L activation at pH 5.5.

In sum, the precision of the kinetic assay demonstrates that this assayis valid for detection of potential cathepsin L activity of adalimumabprocess intermediates.

This application is related to U.S. Pat. Nos. 6,090,382, 6,258,562, and6,509,015. This application is also related to U.S. patent applicationSer. No. 09/801,185, filed Mar. 7, 2001; U.S. patent application Ser.No. 10/302,356, filed Nov. 22, 2002; U.S. patent application Ser. No.10/163,657, filed Jun. 5, 2002; and U.S. patent application Ser. No.10/133,715, filed Apr. 26, 2002; U.S. patent application Ser. No.10/222,140, filed Aug. 16, 2002; U.S. patent application Ser. No.10/693,233, filed Oct. 24, 2003; U.S. patent application Ser. No.10/622,932, filed Jul. 18, 2003; U.S. patent application Ser. No.10/623,039, filed Jul. 18, 2003; U.S. patent application Ser. No.10/623,076, filed Jul. 18, 2003; U.S. patent application Ser. No.10/623,065, filed Jul. 18, 2003; U.S. patent application Ser. No.10/622,928, filed Jul. 18, 2003; U.S. patent application Ser. No.10/623,075, filed Jul. 18, 2003; U.S. patent application Ser. No.10/623,035, filed Jul. 18, 2003; U.S. patent application Ser. No.10/622,683, filed Jul. 18, 2003; U.S. patent application Ser. No.10/622,205, filed Jul. 18, 2003; U.S. patent application Ser. No.10/622,210, filed Jul. 18, 2003; and U.S. patent application Ser. No.10/623,318, filed Jul. 18, 2003. This application is also related toPCT/US05/12007, filed Apr. 11, 2005. The entire contents of each ofthese patents and patent applications are hereby incorporated herein byreference.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims. The contents of allreferences, patents and published patent applications cited throughoutthis application are incorporated herein by reference.

What is claimed:
 1. A method for preparing an adalimumab formulation,comprising the step of mixing a liquid composition comprising adalimumabwith a pharmaceutically acceptable carrier, wherein the adalimumab isexpressed in a Chinese Hamster Ovary (CHO) cell expression system, andthe composition is characterized in that when the composition is assayedin a cathepsin L kinetic assay, a level of cathepsin L activity of lessthan 1.84 RFU/s/mg of adalimumab is observed, wherein the cathepsin Lkinetic assay comprises: i) diluting the composition in a polystyrenecontainer in a solution containing 25 mM NaOAc, 5 mM DTT and 1 mM EDTAat pH 5.5, ii) adding dextran sulfate to a concentration of 0.035 μg/mLand incubating at 37° C. for six hours, iii) adding Z-leucine-argininecovalently bound at its C-terminus to a fluorescent 7-amino-4-methylcoumarin (Z-leucine-arginine-AMC), wherein the diluting, adding, andincubating steps are sufficient to permit the measurement of cathepsin Lhydrolysis of the Z-leucine-arginine-AMC within a linear range, and iv)measuring Z-leucine-arginine-AMC hydrolysis in the linear range inRFU/s/mg of adalimumab.
 2. The method of claim 1, wherein the cathepsinL activity is from about 0.4 to 1.0 RFU/s/mg of adalimumab.
 3. Themethod of claim 1, wherein the cathepsin L activity is from about 0.4 to1.3 RFU/s/mg of adalimumab.
 4. The method of claim 1, wherein thecathepsin L activity is from about 0.4 to 0.6 RFU/s/mg of adalimumab. 5.The method of claim 1, wherein the cathepsin L activity is from about0.4 to 0.85 RFU/s/mg of adalimumab.
 6. The method of claim 1, whereinthe cathepsin L activity is from about 0.4 to 0.9 RFU/s/mg ofadalimumab.
 7. The method of claim 1, wherein the cathepsin L activityis from 0.5 to 1.5 RFU/s/mg of adalimumab.
 8. The method of claim 1,wherein the cathepsin L activity is from 0.4 to less than 1.84 RFU/s/mgof adalimumab.
 9. The method of claim 1, wherein the formulation ispackaged in a pre-filled syringe.
 10. The method of claim 1, wherein theformulation comprises 50 mg/ml of adalimumab.
 11. The method of claim 1,wherein the formulation is suitable for subcutaneous injection.
 12. Themethod of claim 1, wherein the diluted composition has an adalimumabconcentration of 20 μg/ml.
 13. The method of claim 1, wherein thediluted composition has an adalimumab concentration of 50 μg/ml.
 14. Themethod of claim 1, wherein step i) of the cathepsin L kinetic assaycomprises diluting the composition 600 fold.
 15. The method of claim 1,wherein the pharmaceutically acceptable carrier comprises mannitol. 16.The method of claim 1, wherein the pharmaceutically acceptable carriercomprises sodium chloride.
 17. The method of claim 8, wherein theformulation is packaged in a pre-filled syringe.
 18. The method of claim8, wherein the formulation comprises 50 mg/ml of adalimumab.
 19. Themethod of claim 8, wherein the formulation is suitable for subcutaneousinjection.
 20. The method of claim 8, wherein the diluted compositionhas an adalimumab concentration of 20 μg/ml.
 21. The method of claim 8,wherein the diluted composition has an adalimumab concentration of 50μg/ml.
 22. The method of claim 8, wherein step i) of the cathepsin Lkinetic assay comprises diluting the composition 600 fold.
 23. Themethod of claim 8, wherein the pharmaceutically acceptable carriercomprises mannitol.
 24. The method of claim 8, wherein thepharmaceutically acceptable carrier comprises sodium chloride.
 25. Anadalimumab formulation prepared according to the method of claim
 1. 26.An adalimumab formulation prepared according to the method of claim 8.27. A method of treating a disorder in which TNFα activity isdetrimental in a subject, the method comprising administering to thesubject a therapeutically effective amount of the adalimumab formulationof claim 25 such that the disorder is treated, wherein the disorder isselected from the group consisting of rheumatoid arthritis, Crohn'sdisease, ulcerative colitis, ankylosing spondylitis, psoriaticarthritis, psoriasis, and juvenile rheumatoid arthritis.
 28. A method oftreating a disorder in which TNFα activity is detrimental in a subject,the method comprising administering to the subject a therapeuticallyeffective amount of the adalimumab formulation of claim 26 such that thedisorder is treated, wherein the disorder is selected from the groupconsisting of rheumatoid arthritis, Crohn's disease, ulcerative colitis,ankylosing spondylitis, psoriatic arthritis, psoriasis, and juvenilerheumatoid arthritis.